Immunogenic compositions and vaccines comprising carrier bacteria that secrete antigens

ABSTRACT

Disclosed are vaccines and immunogenic compositions which use live attenuated pathogenic bacteria, such as Salmonella, to deliver ectopic antigens to the mucosal immune system of vertebrates. The attenuated pathogenic bacteria are engineered to secrete the antigen into the periplasmic space of the bacteria or into the environment surrounding the bacteria. The vertebrate mounts a Th2-mediated immune response toward the secreted antigen.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application No. 60/372,710 filed Apr. 15, 2002.

GOVERNMENTAL SUPPORT

[0002] This work was supported by the National Institutes of Health grant number DE 06669. The government of the United States of America has certain rights in this invention.

SEQUENCE LISTING

[0003] A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f).

BACKGROUND OF THE INVENTION

[0004] 1. Field of the Invention

[0005] The invention relates generally to vaccines or immunogenic compositions comprising live attenuated bacteria used as carriers to deliver antigens to a vertebrate, wherein an immune response, especially a Th2-mediated immune response, is elicited by the vertebrate toward the antigen.

[0006] 2. Description of the Related Art

[0007] Citations to some of the related art documents may be indicated as numbers in parentheses. Those numbered citations refer to the bibliography that is found at the end of this section. Those related art documents, as well as other references cited throughout this application, are herein incorporated by reference. The inclusion of those related art documents in this application is not an admission that those documents constitute prior art.

[0008] Approximately one-third of the 55 to 60 million human deaths each year are the result of infectious diseases caused by a diversity of pathogens including bacteria, fungi, protozoa, parasites (such as helminths) and viruses. Most of these pathogens are unicellular, although some fungi and parasites are multicellular. Viral pathogens are unable to multiply outside of living hosts and are composed of nucleic acids, polypeptides and sometimes lipids and carbohydrates. Infections by pathogens also cause significant morbidity associated with malnutrition and retarded development especially in children, and suboptimal work performance or absences from work of afflicted adults. The social and economic consequences of infectious diseases are staggering and difficult to compute. It is becoming increasingly clear that many disease states such as ulcers, arthritis, diabetes, cardiovascular illness and cancer may be caused in part by pathogens. Prevention of infectious diseases and immunotherapeutic treatments of those suffering from infectious diseases, including infections that cause ulcers (Helicobacter pylori), hepatomas (hepatitis virus), hardening of the arteries (Chlamydia pneumoniae), via the development of vaccines would contribute significantly to the length and quality of life, and to a significant reduction of the world's wealth used to provide health care to those afflicted with infectious diseases caused by pathogens.

[0009] Many vaccines, especially those containing purified antigenic components or conjugates thereof, are too expensive to have wide utility in the developing world, where economic resources are at a minimum. Such vaccines require refrigeration to maintain potency, which is difficult to provide in tropical parts of the world, and must be administered with syringes that are expensive and require sterilization. Thus, the development of vaccines to be delivered via the mucosal immune system using live carrier bacteria is very important to the public health of the world.

[0010] Bacterial Carriers

[0011] The use of carrier bacteria, including both gram-negative and gram-positive strains, to deliver vaccine antigens to the mucosal immune system is known to be effective in eliciting cellular and humoral immune responses in both the systemic and mucosal immune systems. Attenuated mucosal pathogenic bacteria, such as Listeria monocytogenes, Salmonella spp., Vibrio cholera, Shigella spp., Mycobacterium bovis BCG, Yersinia enterocolitica and Bacillus anthracis, as well as commensal strains of bacteria, such as Streptococcus gordonii, Lactobacillus spp., Staphylococcus spp., and E. coli have been successfully employed as antigen delivery carriers. For a recent review on the art of using live bacterial carriers, see Medina and Guzman, Vaccine 19:1573-1580 (2001) and Curtiss, Clin. Invest. 110:1061-1066 and references cited therein, which are herein incorporated by reference.

[0012] The ability of live recombinant bacteria-based vaccines to colonize the gut-associated lymphoid tissue (Peyer's patches) and the deep tissues following oral administration is beneficial in that it stimulates all arms of the immune response, including mucosal, humoral and cellular immunities (12, 16, 33). Recombinant Salmonella vaccines have also been developed as multivalent vaccines to deliver foreign antigens originating from viruses, bacteria and parasites (12, 33). Orally administered S. typhimurium colonizes the gut-associated lymphoid tissue (Peyer's patches) and the secondary lymphatic tissues including the liver and spleen, to elicit anti-Salmonella immune responses during infection of the mouse (21). The immune responsiveness to orally administered Salmonella has been applied to develop live attenuated oral Salmonella vaccines (12). Attenuated Salmonella vaccines have been constructed by introduction of mutations in the genes required for virulence, including the cyclic AMP receptor protein gene (crp) (11). Crp is a global regulator involved in a variety of biological functions including carbohydrate utilization (4). Attenuated Salmonella vaccine strains have been genetically modified to express heterologous antigens, i.e., antigens that are normally expressed by a different organism, specified by multicopy plasmids. These recombinant vaccines induce immunity to the pathogen whose antigen gene is expressed as well as to Salmonella. It is essential that the antigen specifying plasmids in Salmonella vaccines are stably maintained during the in vivo colonization process. A “balanced-lethal host-vector system” based on the essential bacterial gene aspartate β-semialdehyde dehydrogenase (asd) has been used to specify recombinant antigens from Asd+plasmids that are retained in vivo in asd gene deleted Salmonella vaccine strains (15, 35).

[0013] Analysis of convalescent sera from patients or animals infected with bacterial pathogens reveal that the proteins located in the envelopes of or secreted by the bacterial pathogens act as dominant immunogens for the immune responses (30, 37, 56). These observations indicate that envelope and secreted proteins are highly immunogenic and/or more readily interact with antigen presenting cells (APCs) due to their subcellular location. Translocation of such highly immunogenic antigens into the cell envelope or secretion from the cell should increase the strength of the immune response elicited by vaccine strains expressing foreign antigens. In the development of attenuated Salmonella-based multivalent vaccines, a preferable system would use a foreign antigen secreted from the cytoplasm of Salmonella vaccines (18, 19). β-lactamase, encoded by the ampicillin resistance gene, and hemolysin are well-characterized periplasmic secreted proteins in gram-negative bacteria (41, Su et al., 1992, Microb. Pathog. 13:465-476). It is well known that β-lactamase is secreted into the periplasmic space of gram-negative bacteria and its translocation depends upon the presence of a signal sequence consisting of 23 amino acid residues at the N-terminus (25, 41). Evidence obtained from other studies confirms that the signal sequence plus an additional 12 amino acids of the mature β-lactamase are required to translocate β-lactamase through the cytoplasmic membrane of gram-negative bacteria (27, 51). Fusion of a protein to the β-lactamase signal sequence promotes the secretion of the fusion protein into the periplasm of E. coli (40, 51).

[0014] Immune Responses

[0015] A successfully efficacious vaccine may require that a correct balance is achieved between the different arms of the immune system when an immune response is elicited. Broadly speaking, those arms involve cell-mediated immunity and humoral immunity, which are regulated by helper T cells. Helper T cell-based immune responses are divided into three classes: TH1-, TH2-, and TH0-mediated immune responses. TH1 cells (a) direct cell-mediated immunity, (b) secrete the proinflammatory cytokines gamma-interferon, tumor necrosis factor-beta and lymphotoxin-alpha, and (3) promote class switching to IgG2a. TH1-mediated immunity is generally directed toward intracellular pathogens, including viruses and intracellular bacteria such as Mycobacterium spp. and Salmonella spp. TH1-cells are also involved in inflammation and organ-specific autoimmunity. TH2 cells (a) direct humoral immunity, (b) secrete pro-B-cell cytokines including IL-4, IL-5, IL-9, IL-10, IL-13 and (c) promote class switching to IgG1 and IgA (38, 49). TH2-mediated immunity is generally directed toward extracellular pathogens, including bacteria, helminths and other parasites. TH2-cells are also involved in allergic reactions. TH2-type immune responses are rare in the immune response elicited by attenuated Salmonella vaccines. TH0-cells secrete a combination of TH1 and TH2 cytokines. To date, the mechanism determining TH1- or TH2-type immunity to a given antigen is not well understood. Reviews on TH1- and TH2-mediated immune responses can be found in Golding and Scott, 1995, Ann NY Acad Sci, 754:126-137, Del Prete, G., 1992, Allergy, 47:450-455, and Dong and Flavell, 2000, Arthritis Res, 2:179-188, which are herein incorporated by reference.

[0016] Vaccine Directed Against Pneumococcal Antigens

[0017]Streptococcus pneumoniae is a human pathogen that causes life-threatening diseases, including community-acquired pneumonia, otitis media, meningitis, and bacteremia in persons of all ages (34). Pneumococcal pneumonia is the leading cause of childhood death worldwide, resulting in over 3 million deaths per year (20). The recent emergence of antibiotic resistant strains has the potential to threaten the treatment of pneumococcal disease in the near future (5). Thus the development of a cost effective pneumococcal vaccine is urgent. Capsular polysaccharide-based pneumococcal vaccines are currently available and are moderately effective. A 23-valent pneumococcal polysaccharide vaccine is recommended for the prevention of infection in adults (46) and a 7-valent conjugated polysaccharide vaccine is licensed for use in children (47). However, vaccination with the pneumococcal polysaccharide vaccine does not reduce the frequency of hospitalization, costs, and mortality caused by pneumococcal pneumonia (22), which reinforces the need for effective new vaccines.

[0018] Studies of the protective efficacy of subunit vaccines may further the development of a more protective pneumococcal vaccine. The pneumococcal surface protein A (“PspA”) has been evaluated and considered a pneumococcal vaccine candidate because of its immunogenicity and protection of mice against virulent S. pneumoniae challenge (6, 8, 9, 24). Native PspA_(RX1) (PspA originating from S. pneumoniae Rx1 strain) contains several functional domains: amino terminal signal sequence, alpha-helical region, a proline-rich domain, 10 tandem-repeat choline-binding regions and a 17-amino acid residue carboxy terminus. Pneumococcal protection assays in mice immunized with various recombinant PspA_(RX1) oligopeptides showed that the alpha-helical domain contains the protective epitopes (7). In another study, mice orally immunized with a S. typhimurium vaccine strain expressing a recombinant PspA_(RX1) (from ATG start codon through signal sequence up to the fifth tandem repeat) elicited PspA-specific immune responses and protected against virulent S. pneumoniae challenges (36). Expression of recombinant PspA in this recombinant Salmonella vaccine strain was somewhat toxic such that the high copy number plasmid pYA3193 (pUCori) specifying PspA was relatively unstable. Thus, approximately 50% of cells lost the plasmid after 24 h growth as a standing culture, which is unacceptable for a vaccine or immunogenic composition.

CITATIONS TO RELATED ART

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SUMMARY OF THE INVENTION

[0076] The inventors have discovered that antigens, which are secreted from a bacterial cell into a vertebrate host cell, stimulate in vertebrates a TH2-type immune response, as ascertained by the detection of IgG1 or IgA antibodies that bind to the secreted antigen, directed toward the secreted antigens. Thus, the present invention is directed to an improved bacteria-based vaccine comprising a plasmid vector that (a) enables the stable expression and secretion of recombinant pathogen antigens, such as PspA, (b) induces a higher immune response to the recombinant pathogen antigen than to endogenous antigens that are expressed by the carrier bacteria, as determined by quantitative analysis of specific antibodies produced by the host vertebrate, and (c) stimulates a TH2-type immune response in the recipient vertebrate. The present invention is drawn to live attenuated pathogenic bacteria (“the carrier bacteria”) that are useful as vaccine carriers, and vaccines and immunogenic compositions that comprise a carrier bacteria. Preferably the bacteria are gram negative bacteria, especially members of the Enterobacteriaceae, Vibrionaceae, Francisellaceae, Legionallales, Pseudomonadacea or Pasteurellaceae groups, including Salmonella spp., Shigella spp., Escherichia spp., Yersinia spp., and Vibrio spp., and the antigen is an antigen derived from a pathogen that is different than the particular strain of carrier bacteria that is used. However, the carrier bacteria may be any bacteria that can infect any one or more of many mucosal membranes, including gut, bronchial, alveolar, conjunctival, and nasal mucous membranes. The carrier bacteria may contain a genetic mutation that results in attenuating the virulence of the carrier bacteria. Attenuating mutations include mutations in the gene encoding cyclic AMP receptor protein (Crp), e.g., Δcrp-28, the gene encoding DNA adenine methylase (Dam), the gene encoding adenylate cyclase (Cya), and the gene encoding β-semialdehyde dehydrogenase (Asd), among others, including for example mutations in the aroA, aroC, aroD, purA, purB, purE, cdt, phoP, phoQ, hemA, ompR, ompc, ompF, rpoS, fur, rfa, rfb, rfc, pmi, galE, and htrA genes.

[0077] The carrier bacteria contains a polynucleotide encoding an antigen, which is derived from another pathogen, wherein the antigen is fused to a signal sequence or other secretion peptide, which targets the antigen for secretion from the cytoplasm of the carrier bacteria. In another embodiment, the carrier bacteria may contain two or more polynucleotides, each of which encode an antigen from a different serotype of the same pathogen (such as the PspA antigen from the EF5668 strain and the Rx1 strain of Streptococcus pneumoniae), which are fused to a signal sequence. An object of this multiple antigen embodiment is to provide broad immunological protection to the majority of serotypes of a given pathogen. The antigen (or multiple antigens) is secreted from the bacterial cell into the periplasmic space of the bacterium or into the surrounding environment, which may be the interior of the host vertebrate cell or the interstitial space outside of the host cell, but within the host tissues. When the vaccine, immunogenic composition or carrier bacteria is administered to a vertebrate, the antigen is secreted from the carrier bacteria and the vertebrate elicits a Th2 response toward the secreted antigen. Preferred vertebrates are humans, livestock, poultry, and companion animals. Preferred pathogens from which the antigen is derived, especially when the vertebrate is a human, include Streptococcus pneumoniae., Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Erysipelothrix rhusiopathiae, Mycobacterium tuberculosis, Listeria spp., Bacillus anthracis, Clostridium spp., and Corynebacterium spp. The preferred antigen is a pneumococcal surface protein A (“PspA”), which sequence is set forth in SEQ ID NO:1 or SEQ ID NO:26, or a fragment of SEQ ID NO:28. The preferred secretion peptide is a fragment of the β-lactamase enzyme, which fragment sequence is set forth in any one of SEQ ID NO:2 or SEQ ID NO:3, or a portion of SEQ ID NO:28.

[0078] The carrier bacteria may comprise a balanced-lethal host system to help maintain the polypeptide, which encodes the secretion competent antigen, in the carrier bacteria. The preferred balanced lethal host system relies on the complementation of a lethal mutation (ΔasdA16) in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd). Asd is required for the synthesis of diaminopimelic acid (“DAP”), an essential component of the rigid layer of the bacterial cell wall. The polynucleotide encoding the secretion competent antigen is placed onto a plasmid that contains a functional copy of a gene encoding Asd. Thus, in order for the carrier bacteria to survive, it must maintain the plasmid, which contains the polynucleotide encoding the secreted antigen.

[0079] The carrier bacteria may comprise an environmental limitation viability system, which ensures that the carrier bacteria will not survive in the environment outside of the vertebrate to which the carrier bacteria was administered. For example, the carrier bacteria may contain a mutation in an essential gene, wherein the essential gene is complemented by a regulatable copy of the essential gene, such that a functional gene product is expressed only at the temperature found within a vertebrate; or the carrier bacteria may contain a gene that encodes a polypeptide that is toxic to the carrier bacteria, wherein the toxic polypeptide is produced only at the temperature outside of the vertebrate. Alternative biological containment systems are described in U.S. provisional patent application serial No. 60/407,522 and U.S. patent application entitled “Regulated Attenuation of Live Vaccines to Enhance Cross-Protective Immunogenicity. Filed Apr. 15, 2003, both of which are hereby incorporated by reference.

[0080] The invention is also drawn to methods of eliciting an immune response in a vertebrate, wherein the carrier bacteria, as described in the preceding paragraphs, is administered to the vertebrate. The antigen, which is derived from a pathogen that is different than the carrier bacteria, is secreted from the carrier bacteria and the vertebrate produces IgG1 antibodies that specifically bind to the antigen. Preferably the vertebrate is a human, livestock, poultry or companion pet. Preferably the antigen is derived from a pathogen such as Streptococcus pneumoniae, Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Erysipelothrix rhusiopathiae, Mycobacterium tuberculosis, Listeria spp., Bacillus anthracis, Clostridium spp., and Corynebacterium spp.

BRIEF DESCRIPTION OF THE DRAWINGS

[0081]FIG. 1: Reduced expression of Asd protein by deletion of asd gene promoter region. SDS-PAGE was performed with cell lysates of S. typhimurium×4550 with pYA3333 (entire asd gene with P_(asd), pBRori), pYA3334 (entire asd gene P_(asd), pUCori), pYA3342 (promoterless SD-asd gene, pBRori) and pYA3341 (promoterless SD-asd gene, pUCori). Standards are indicated to the left and Asd protein (39 kDa) is designated by arrow. Lanes; 1, pYA3333; 2, pYA3334; 3, pYA3342; 4, pYA3341.

[0082]FIG. 2: Asd⁺ antigen expression vectors. (A) Asd⁺ vector pYA3342. The map of pYA3342 and the nucleotide sequences of the P_(trc), promoter region and multicloning sites are depicted. (B) Periplasmic secretion Asd⁺ vector pYA3493. A DNA fragment encoding the β-lactamase signal sequence and 12 amino acid residues of the N-terminus of mature β-lactamase of plasmid pBR322 (SEQ ID NO:2) was positioned under the control of the P_(trc) promoter of an Asd⁺ vector pYA3342 (pBRori). The map of pYA3493 and the nucleotide sequences of the P_(trc) promoter region, β-lactamase signal sequence (bla SS) and multicloning sites are depicted. The P_(trc) sequences for -35, -10, Shine-Dalgarno (SD), and translation start codon are bold typed. An arrow within the sequence indicates the signal peptidase cleavage site. Unique restriction enzyme sites in the multicloning site are indicated. 5ST1T2 is a transcriptional terminator.

[0083]FIG. 3: Recombinant plasmid pYA3494 for PspA overexpression. (A) PspA region used in this study. Functional domains of native PspA from S. pneumoniae (PspA) are diagramed; dotted box, leader sequence (31 aa); open box, immuno dominant a-helical region (1-288 aa); box with slanted lines, proline-rich region (289-370 aa); ten gray boxes, choline-binding repeats (371-571 aa); black box, C-terminus (572-588 aa). Dotted lines represent the limit of the recombinant PspA (rPspA) region used in this study. Bioinformatical analyses of the PspA for antigenic index and surface probability are presented. Analyses were performed with the Protean module of the Lasergene sequence analysis software. (B) The map of recombinant plasmid pYA3494. A 0.7 kb EcoRI-Hind III fragment of PCR amplified DNA fragment of pspA_(rx1) was cloned into the EcoRI and HindIII sites of pYA3493 (FIG. 2B). The cloned fragment included the immunogenic α-helical region of PspA including amino acids 3 through 257 of mature PspA (255 amino acids).

[0084]FIG. 4: Subcellular location of expressed rPspA in S. typhimurium. Subcellular fractions were prepared from S. typhimurium×8599 (pYA3494) cells grown in LB broth at 37° C. Fractions equivalent to 30 μl volume of 0.8 OD₆₀₀ culture except for supernatant fluid were analyzed by SDS-PAGE and the rPspA was detected by immunoblot with PspA specific monoclonal antibody Xi126. β-galactosidase and OmpC were used as fractionation controls for cytoplasmic and outer membrane fractions, respectively. Standards are indicated to the left. Lanes; 1, total cell lysate; 2, cytoplasm; 3, periplasm; 4, outer membrane; 5, concentrated supernatant (750 μl); 6, supernatant (10 μl).

[0085]FIG. 5: Expression of rPspA in the S. typhimurium vaccine strain. ×8501 harboring pYA3494 (specifying rPspA) or pYA3493 (vector control) was cultured in LB broth at 37° C. Total cells (equivalent to 7.5×10⁸ cells) and concentrated culture supernatants (equivalent to 750 μl of supernatant of 0.8 OD₆₀₀ culture) were subjected to SDS-PAGE analysis. The left panel is a Coomassie brilliant blue stained gel. The right panel is an immunoblot of the duplicated gel with PspA specific monoclonal antibody Xil26. Molecular markers are indicated to the left. PspA proteins are indicated. Lanes 1 and 2 represent protein profiles of ×8501 (pYA3493) and ×8501 (pYA3494), respectively.

[0086]FIG. 6: Serum IgG responses to S. typhimurium LPS and SOMPs, and foreign antigen rPspA. The data represent IgG antibody levels, as determined by ELISA, induced in mice orally immunized with ×8501 (pYA3493) (vector control) and ×8501 (pYA3494) (expressing rPspA) at designated weeks after immunization. Black arrows indicate sublethal i.v, infection with S. pneumoniae WU2. Columns; 1, 2 weeks; 2, 4 weeks; 3, 6 weeks; 4, 8 weeks; 5, 10 weeks; 6, 12 weeks; 7, 17 weeks; 8, 19 weeks, 9, 21 weeks.

[0087]FIG. 7: Secretory IgA responses to S. typhimurium LPS and SOMPs, and recombinant PspA. The data represent anti-LPS, -SOMPs and -rPspA IgA antibody levels in vaginal secretions of BALB/c mice orally immunized with ×8501 (pYA3493) (vector control) and ×8501 (pYA3494) (expressing rPspA) at weeks 4, 6, 8, and 10 after immunization.

[0088]FIG. 8. Serum IgG2a and IgG1 responses to S. typhimurium LPS and SOMPs, and recombinant PspA. The data represent IgG2a and IgG1 subclass antibody levels to Salmonella LPS and SOMPs and rPspA in sera of BALB/c mice orally immunized with ×8501 (pYA3493) (vector control) and ×8501 (pYA3494) (expressing rPspA) at designated weeks after immunization. Black arrows indicate sublethal i.v. infection with S. pneumoniae WU2. Anti-rPspA IgG2a and IgG1 responses of ×8501 (pYA3493) (negative control) were not shown. Columns; 1, 2 weeks; 2, 4 weeks; 3, 6 weeks; 4, 8 weeks; 5, 10 weeks; 6, 12 weeks; 7, 17 weeks; 8, 19 weeks; 9, 21 weeks.

[0089]FIG. 9. Relationship between PspA amino acid sequences. This figure diagrammatically depicts the familial relationship among the PspA proteins in S. pneumoniae of diverse capsular polysaccharide serotypes.

[0090]FIG. 10 depicts the nucleotide sequence (SEQ ID NO:13) and protein sequence (SEQ ID NO:26) for the N-terminal portion of the EF5668 pspA gene including the signal sequence and the α-helical domain.

[0091]FIG. 11 lists all the oligonucleotide primers to first clone the EF5668 α-helical domain into pYA3493 to yield pYA3594. Primers 1 through 6 are SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively.

[0092]FIG. 12 depicts the stepwise construction of plasmid pYA3605.

[0093]FIG. 13 depicts the polynucleotide encoding the β-lactamase/PspA_(EF5668)/PspA_(Rx1) fusion protein (SEQ ID NO:20), and the conceptually translated polypeptide sequence (SEQ ID NO:28).

[0094]FIG. 14. Western blot analysis of β-lactamase/PspA_(EF566)/PSPA_(Rx1) fusion protein expression by ×6212. Monoclonal antibodies directed against both PspA_(EF5668) and PspA_(Rx1) antigens were each able to detect the fusion protein, which was of the expected size.

[0095]FIG. 15 depicts the stepwise construction of plasmid pYA3620.

[0096]FIG. 16 depicts the polynucleotide sequence of β-lactamase signal sequence (bla SS) and carboxyterminal (bla CT) sequence as in plasmid pYA3620 (SEQ ID NO:25), and the corresponding conceptually translated polypeptide sequences.

[0097]FIG. 17 depicts the IgG immune responses to LPS and PspA induced by Salmonella vaccine strain ×8501 comprising pYA3494, which encodes secretion competent PspA, and Salmonella vaccine strain ×8501 comprising pYA3496, which encodes cytoplasmic PspA.

[0098]FIG. 18 shows the pspA sequence from S. pneumoniae Rx1.

[0099]FIG. 19 shows the DNA and amino acid sequences of the codon optimized pSpA_(Rx1) with optimized codons indicated in bold-face type.

[0100]FIG. 20 shows the DNA and amino acid sequences of the codon optimized pSPA_(EF5668) with optimized codons indicated in bold-face type.

[0101]FIG. 21 shows the DNA and amino acid sequence of the codon optimized pspA_(EF5668-Rx1) fusion with optimized codons indicated in bold-face type.

[0102]FIG. 22 is a list of primer used for constructions shown in FIGS. 19, 20 and 23.

[0103]FIG. 23 illustrates contruction of pspA plasmids using oligonucleotide sequences shown in FIG. 22.

[0104]FIG. 24 shows vectors with original pneumococcal and codon optimized sequences for PSPA_(Rx1), PSPA_(EF5668) and the PspA_(EF5668-Rx1) fusion.

[0105]FIG. 25 illustrates construction of pYA3637 with the codon optimized PSPA_(EF5668) sequence inserted into the pYA3620 expression vector.

[0106]FIG. 26 shows the DNA and amino acid sequences of the bla SS-pspA_(EF5668)-bla C-terminus region in pYA3637.

[0107]FIG. 27 illustrates the expression of PspA_(EF5668), PspA_(Rx1) and fPspA (PspA_(Ef5668-Rx1)) in Salmonella.

[0108]FIG. 28 shows the pYA3332 Asd⁺ vector with pl5A ori.

[0109]FIG. 29 shows Salmonella chromosomal insertion-deletion and deletion mutations.

[0110]FIG. 30 shows suicide vectors for introducing the ΔilvG3::TT araC P_(BAD)/ac/TT, ΔaraBAD23, and ΔaraE25 mutations.

[0111]FIG. 31 shows the DNA and amino acid sequences of pspA from S. pneumoniae 6B.

DETAILED DESCRIPTION OF THE INVENTION

[0112] The invention is directed to an improved vaccine or immunogenic composition that delivers an antigen to the mucosal immune system of a vertebrate and provokes a Th2-mediated immune response in the recipient vertebrate against the antigen. The invention is also directed to methods of eliciting a Th2-mediated immune response in a vertebrate, comprising administering a vaccine or immunogenic composition to the vertebrate. The vaccine or immunogenic composition comprises a live attenuated strain of pathogenic bacteria, wherein the strain of bacteria is capable of infecting or colonizing any one or more mucosal tissues within the vertebrate. As used herein, the term “attenuated” means that the carrier bacteria is less virulent than the wild-type version of the bacteria from which the carrier is derived. The term “virulence” means the ability of a pathogen to cause disease. Preferred vertebrates include humans, livestock, poultry and companion pets.

[0113] The mucosal immune system comprises several components, including the nasal-associated lymphoid tissue (“NALT”), bronchus-associated lymphoid tissue (“BALT”), gut-associated lymphoid tissue (“GALT”), intestinal epithelial cells (“IEC”), lamina propria, dendritic cells, mucosal macrophages, and the like. For a detailed discussion of the mucosal immune system, see Mucosal Immunology 2^(nd) edition, edited by Ogra, P. L. et al., Academic Press, San Diego, 1999, which is herein incorporated by reference.

[0114] The improved vaccine or immunogenic composition comprises a live attenuated strain of a pathogenic bacteria capable of infecting any one component or several components of the mucosal immune system. The live attenuated strain of pathogenic bacteria, also referred to as the “carrier”, contains a polynucleotide that encodes an antigen derived from a pathogen that is different than the carrier bacteria, wherein the antigen is secreted from the carrier bacteria. The carrier bacteria may also comprise multiple polynucleotides, each of which encodes an antigen from a different serotype of the same pathogen (that is different than the carrier bacteria). As used herein, the term “serotype” or the phrase “different serotype of the same pathogen” means a closely related strain, usually a strain of bacteria, found within the same species. For example, Salmonella typhi and Salmonella typhimurium are different serotypes of the species Salmonella enterica. In another example, Streptococcus pneumoniae strain EF5668 and Streptococcus pneumoniae strain Rx1 are different serotypes of the species Streptococcus pneumoniae. The term “serotype” is a term of art.

[0115] Methods of making and using live attenuated strains of bacteria that are suitable for vaccines or immunogenic compositions, including instruction on how to make mutations in virulence genes, are taught in U.S. Pat. Nos. 5,294,441, 5,387,744, 5,389,368, 5,468,485, 5,855,879, and 5,855,880, which are herein incorporated by reference. Those patents teach methods of attenuating pathogenic bacteria to be used as vaccine or immunogenic composition carriers. Examples of genes that may be mutated to confer attenuation include cya (which encodes adenylate cyclase), crp (which encode cAMP receptor protein), asd and dam (which encodes DNA adenine methylase). The role of mutations in the dam gene is discussed in Fricker, J., “New vaccines: damming for multi-strain organisms?” Drug Discovery Today 7:212-213 (2002) and all references cited therein, which are herein incorporated by reference. In a preferred embodiment of the invention, the live attenuated carrier bacteria is rendered attenuated by having an inactivating mutation in the crp gene.

[0116] The vaccine, immunogenic composition or carrier bacteria may comprise a “balanced-lethal host-vector system”, which enables the carrier bacteria to maintain the presence of the ectopic polynucleotide constructs without the need for external selection. How to make and use the “balanced-lethal host-vector system” are taught in U.S. Pat. Nos. 5,294,441, 5,387,744, 5,424,065, 5,656,488, 5,672,345, 5,840,483, 5,855,879, 5,855,880 and 6,024,961, and PCT/US01/13915, which are herein incorporated by reference. The balanced-lethal host-vector system is based upon the concept of having an inactivating mutation in an essential gene of the carrier bacteria, wherein a functional copy of the essential gene is provided on a plasmid (a genetically engineered autonomous extrachromosomal polynucleotide), which contains the polynucleotide that encodes the antigen. Thus, in order for the carrier bacterium to remain viable, the plasmid, which contains the functional copy of the essential gene and the polynucleotide that encodes the antigen, must be maintained in the carrier bacterium. This ensures that the polynucleotide that encodes the antigen is maintained in the carrier bacterium. In a preferred embodiment of the invention, the essential gene encodes β-aspartate semialdehyde dehydrogenase (Asd), and the plasmid encodes a functional Asd polypeptide, which complements the chromosomal asd mutation, but which cannot replace the defective chromosomal Asd gene by recombination. Lack of a functional Asd polypeptide causes bacterial cells to lyse. The polynucleotide encoding the functional Asd polypeptide and the polypeptide that encodes the antigen are physically linked on the same episome, thereby ensuring that the carrier bacteria maintains the polynucleotide that encodes the antigen.

[0117] The vaccine or immunogenic composition may also comprise an “environmental limited viability system” to prevent the survival of, or to kill those carrier bacteria that escape into the environment. How to make and use the “environmental limited vaccine system” are taught in copending U.S. patent applications Ser. Nos. 08/473,789 and 08/761,769, which are herein incorporated by reference. Alternative biological containment systems are described in U.S. provisional patent application serial No. 60/407,522 and U.S. patent application entitled “Regulated Attenuation of Live Vaccines to Enhance Cross-Protective Immunogenicity,” filed Apr. 15, 2003, both of which are hereby incorporated by reference.

[0118] The inventors have discovered that a Th2-mediated immune response is elicited toward antigens that are secreted by the carrier bacteria, as compared to antigens that are not secreted by the carrier bacteria. To allow for the secretion of the antigen from the carrier, the polynucleotide encoding the antigen is operably linked to a second polynucleotide sequence that encodes a peptide that enables the secretion of the antigen from the carrier. A subset of the peptides that enable the secretion of antigens and other polypeptides from a cell are known in the art as signal sequences. As used herein, the term “secretion peptide” means any peptide, which when fused to any polypeptide facilitates the secretion of that polypeptide from a cell. Any secretion peptide may be used in the practice of the instant invention. In a preferred embodiment of this invention, the secretion peptide comprises a fragment of a β-lactamase polypeptide. Most preferably, the secretion peptide comprises (a) the 35 amino-terminal amino acids of the precursor β-lactamase polypeptide as set forth in SEQ ID NO:2 or (b) the 21 carboxy-terminal amino acids of β-lactamase as set forth in SEQ ID NO:3. However, the skilled artisan in the practice of this invention may substitute any peptide sequence as a secretion peptide, as long as the substitute peptide facilitates or enables the secretion of the antigen from the carrier bacteria, is stable, and is non-toxic to the vertebrate or bacterial host. Preferred secretion peptides are derived from gram negative bacteria.

[0119] Carrier bacteria that are applicable to the operation of this invention may be selected on the basis of their ability to infect or colonize specific body sites. However, it has been demonstrated that gut pathogen-based vaccine carriers, such as attenuated Salmonella, are capable of delivering antigens to several mucosal sites in addition to GALT, including nasal, vaginal, oral and rectal. Therefore, as is shown in the examples that follow, administration to the GALT of an antigen of a respiratory pathogen will result in protective immunity toward the respiratory pathogen. Thus the skilled artisan would reasonably expect the administration of an Enterobacteriaceae carrier, for example, that expresses a respiratory pathogen antigen to be effective in eliciting a protective immune response against the respiratory pathogen.

[0120] Carrier bacteria and antigens are also selected on the basis of the nature of the intended recipient vertebrate. For humans, the carrier bacterium may be a Vibrio cholera, Shigella spp., Yersinia spp., or any one of Salmonella enteritidis serotypes, such as Salmonella typhi or Salmonella paratyphi A, B or C. For animals, the carrier bacteria may be, for example, Salmonella strains such as Salmonella choleraesuis for swine, S. dublin for cattle, S. abortusovis for sheep, or S. gallinarum for poultry. Given the fact that the mucosal vaccination art is replete with methods of delivering antigens to mucosal surfaces, the skilled artisan will readily recognize that other bacterial carriers may be used in the practice of this invention. Strains of bacteria, which may be suitable in the practice of this invention, include Salmonella spp., Vibrio cholerae, Yersinia enterocolitica, Shigella spp., and Escherichia coli, and hybrids thereof. These and other carrier bacteria, as well as pathogens toward which the vaccine or immunogenic composition may be directed, are taught in U.S. Pat. Nos. 4,888,170, 5,110,588, 5,389,368, 5,468,485, 5,888,799 and 6,024,961, and art recognized publications, including Velge-Roussel et al., Infect Immun 68:969-972 (2000), Kyd and Cripps, Vaccine 17:1775-1781 (1999), Husband, Vaccine 11:107-112 (1993), Mestecky and McGhee, Adv Exp Med Biol 327:13-23 (1992), Langermann et al., Nature 372:552-555 (1994), Rush et al., Adv Exp Med Biol 371 B: 1547-1552 (1995), Sizemore et al., Vaccine 15:804-807 (1997), Robinson et al., Nat Biotechnol 15:653-657 (1997), Shaw et al., Immunology 100:510-518 (2000), Thole et al., Curr Opin Mol Ther 2:94-99 (2000), Lee at al., Vaccine 19:3927-3935 (2001), Medina and Guzman, Vaccine 19:1573-1580 (2001), Wells et al., Antonie Van Leeuwenhoek 70:317-330 (1996), which are herein incorporated by reference.

[0121] Pathogens to which the vaccine or immunogenic composition is directed include worms and other helminths, fungi, viruses, protozoans, neoplastic cells, and bacteria. While gametes are not pathogens, it is envisioned that the vaccine or immunogenic composition of the instant invention may be directed toward gametes and thus may be used as a birth control method or anti-fertility treatment. The antigen to be expressed and secreted by the carrier bacteria is selected from the pathogen to which the vaccine or immunogenic composition is directed. For a highly specific or narrow spectrum vaccine or immunogenic composition, the antigen preferably comprises an epitope that is found only in a particular pathogen or serotype thereof. Preferably, to make an effective vaccine, such an epitope is universally recognized. For a general or broad spectrum vaccine or immunogenic composition, the antigen preferably comprises an epitope or group of epitopes that is found in several pathogens or serotypes thereof. Also, broad spectrum vaccines or immunogenic compositions may ectopically express multiple antigens that are shared among different pathogens. Preferred pathogens, include Clostridium spp., Corynebacterium spp., Bacillus anthracis, Streptococcus pneumoniae, Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Streptococcus pyogenes, Erysipelothrix rhusiopathiae, and Mycobacterium tuberculosis.

[0122] In a preferred embodiment of this invention, the secretion-competent antigen comprises a polypeptide fragment of the pneumococcal surface protein A (“PspA”) fused to the β-lactamase signal sequence plus the first 12 amino acids of the secreted from of β-lactamase. The fragment of the PspA may comprise the alpha helical domain of Streptococcus pneumoniae strain EF5688 (SEQ ID NO:1, amino acid residues 110-384 of SEQ ID NO:26), the alpha helical domain of Streptococcus pneumoniae strain Rxl, or both. As discussed in Example 5, the strain EF5688 represents one major family of Streptococcus pneumoniae, while strain Rx1 represents the other major family of Streptococcus pneumoniae (see FIG. 9.) Therefore, the combination of PspA fragments from both strain EF5688 and strain Rx1 is expected to provide an antigen that elicits an antibody immune response that would be protective against infection with the greatest diversity of Streptococcus pneumoniae capsular polysaccharide serotype strains. A preferred secretion-competent antigen comprises a sequence as set forth in SEQ ID NO:25. Other preferred antigens include codon-optimized sequences encoding PspA, as described in Example 8, and an alpha-helical domain of PsaA antigen as described in Example 12.

[0123] The vaccine or immunogenic composition of the instant invention may be administered to vertebrates, which include humans as well as companion pets, vermin, livestock and poultry according to several methods. In order for a vaccine to be effective in stimulating a Th2-mediated immune response, the antigenic materials must be released or presented in such a way to trigger the induction of memory T-cells or other CD4⁺ T-cells directed at the Th2 arm of the immune system of the vaccinated vertebrate. Therefore, the carrier bacteria comprising of the polypeptide that encodes the secretion-competent antigen must be introduced into the vertebrate. In order to stimulate a preferred response of the gut-associated lymphoid tissue (GALT) or bronchus-associated lymphoid tissue (BALT), introduction of the vaccine or immunogenic composition directly into the gut or bronchus is preferred, such as by oral administration, gastric intubation or intranasally in the form of aerosols, although other methods of administering the vaccine, such as intravenous, intramuscular or subcutaneous injection, or intramammary, intrapenial, vaginal or rectal administration, are possible.

[0124] Administration of a live vaccine of the type disclosed above to an individual can be by any known technique. These include oral ingestion, gastric intubation, broncho-nasal-ocular spraying, or whole-body spray. The whole-body spray method of administering a vaccine or immunogenic composition, wherein droplets comprising the attenuated carrier bacteria are delivered as coarse droplets to the body of the recipient vertebrate, is described in detail in the PCT publication WO 00/04920, which is herein incorporated by reference. All of these methods allow the live vaccine to easily reach the GALT or BALT cells and induce antibody formation and cell mediated immunity. Other methods of administration, such as intravenous injection, that allow the carrier microbe to reach the individual's blood stream can be acceptable. Intravenous, intramuscular or intramammary injection are also acceptable with other embodiments of the invention. The immunization dosages required will vary with the antigenicity of the gene product and need only be an amount sufficient to induce an immune response. Routine experimentation will easily establish the required amount. Multiple dosages are used as needed to provide the desired level of protection.

[0125] The pharmaceutical carrier or excipient in which the vaccine is suspended or dissolved may be any solvent or solid or encapsulating material such as for a lyophilized form of the vaccine. The carrier is non-toxic to the vertebrate and compatible with the carrier bacteria and antigenic gene product. Suitable pharmaceutical carriers are known in the art and, for example, include liquid carriers, such as normal saline and other non-toxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose. Gelatin capsules can serve as carriers for lyophilized vaccines. Adjuvants may be added to enhance the antigenicity if desired, but are generally not required to induce an effective immune response, since the bacterial carriers themselves generally serve as adjuvants. When used for administering via the bronchial tubes, the vaccine is preferably presented in the form of an aerosol. Suitable pharmaceutical carriers and adjuvants and the preparation of dosage forms are described in, Remington's Pharmaceutical Sciences, 17th Edition, (Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1985), which is herein incorporated by reference.

[0126] Immunization of a vertebrate with an antigen derived from a pathogen can also be used in conjunction with prior immunization with the carrier bacteria, which expresses the antigen derived from the pathogen. Such parenteral immunization can serve as a booster to enhance expression of the Th2-mediated immune response. The enhanced response is known as a secondary, booster, or anamnestic response and results in prolonged immune protection of the host vertebrate. Booster immunizations may be repeated numerous times with beneficial results.

[0127] The above disclosure describes several preferred embodiments of the invention. The skilled artisan will recognize that other embodiments of this invention, which are not overtly disclosed herein, may be employed in the practice of this invention. The invention is further illustrated by the examples described below, which are not meant to limit the invention.

EXAMPLES

[0128] For ease of reading, citations to related art documents are indicated as numbers in parentheses. Those numbered citations refer to the bibliography in the section “Background of the Invention”, subsection “Citations to related art”. Those related art documents have been incorporated by reference. TABLE 1 Bacterial strains and plasmids Derivation, source or Strain or plasmid Relevant characteristics^(a) reference Strains E. coil χ6212 F φ80d IacZ ΔM15 deoR Δ(IacZYA-argF)U169 supE44 35 λgyrA96 recA1 relA1 endA1 ΔasdA4 Δzhf-2::Tn10 hsdR17(R⁻M⁺) MGN-617 thi-1 thr-1 leuB6 fhuA21 lacY1 gln V44 ΔasdA4 recA1 42 rp4 2-Tc::Mu (λpir), Km^(r) S. typhimurium χ3339 SL1344 hisG 21 χ3761 UK-1 wild-type Curtiss et. al. 1991 χ4550 SR-11 gyrA1816 Δcrp-1 ΔasdA1 Δ(zhf-4::Tn10) Δcya-1 44 χ4746 nadA540::Tn10Δ(galE-ch1-uvrB)-1005, Tet^(r) χ3339 χ8477 UK-1 ΔaraE25 χ3761 χ8449 hisG Δcrp-28 χ3339 χ8501 hisG Δcrp-28 ΔasdA16 χ8449 χ8554 hisG ΔasdA16 χ3339 χ8599 hisG ΔasdA16 atrB13::MudJ χ8554 χ8623 UK-1 ΔilvG3::TT araC P_(BAD) lacl χ3761 χ8767 UK-1 ΔaraBAD23 χ3761 JF2430 LT-2 atrB13::MudJ 14 S. paratyphi A χ8219 wild type, ATCC#9281 ATCC χ8387 Pla χ8219 χ8488 ΔphoP24 χ8387 χ8523 ΔphoP24 ΔasdA16 χ8488 χ8694 ΔasdA25 χ8387 S. typhi χ8438 Ty2 wild-type, RpoS⁺, ATCC#202182 Curtiss collection χ8542 ΔphoP24 χ8438 χ3744 ISP1820 wild-type Curtiss collection χ8521 ΔphoP24 χ3744 χ8522 ΔphoP24 ΔasdA16 χ8521 S. pneumoniae WU2 wild-type virulent, encapsulated type 3, PspA type 1 6 EF5668 Capsular type 4, PspA type 12 McDaniel et. al. 1992 Rx1 Nonencapsulated avirulent, highly transformable variant McDaniel et. al. of D39, PspA type 25 1986 6B Capsular type Sampson et. al. 1997 Plasmids pYA3193 Asd⁺vector harboring 1.5 kb C-terminally truncated 36 pspA gene, pUC ori pYA3332 Asd⁺, p15A ori Curtiss collection pYA3333 Asd⁺, pBR ori Curtiss collection pYA3334 Asd⁺, pUC ori Curtiss collection pYA3341 SD-Asd⁺, pUC ori Curtiss collection pYA3342 SD-Asd⁺, pBR ori Curtiss collection pYA3485 Suicide vector to introduce ΔaraE25 mutation, Cm^(r) Curtiss collection pYA3493 pYA3342 derivative β-lactamase signal sequence- Curtiss collection based periplasmic secretion plasmid pYA3494 0.7 kb DNA encoding the α-helical region of PspA in Curtiss collection pYA3493 pYA3496 0.7 kb DNA encoding the α-helical region of PspA in Curtiss collection pYA3342 for the expression of His-tagged PspA pYA3599 Suicide vector to introduce ΔaraBAD23 mutation, Cm^(r) Curtiss collection pYA3605 pYA3494 derivative harboring pspA_(EF5668-Rx1) Curtiss collection pYA3620 pYA3342 derivative harboring β-lactamase signal Curtiss collection sequence and C-terminal sequence-based periplasmic secretion plasmid pYA3623 pYA3494 derivative harboring pspA_(EF5668) Curtiss collection pYA3633 pYA3494 derivative harboring codon optimized Curtiss collection pspA_(EF5668) sequence pYA3634 Replacing pYA3494 in which base G is deleted at Curtiss collection mature amino acid 234. pYA3635 pYA3494 derivative harboring codon optimized Curtiss collection pspA_(Rx1) sequence pYA3636 pYA3494 derivative harboring codon optimized Curtiss collection pspA_(EF5668-Rx1) sequence pYA3637 pYA3620 derivative harboring codon optimized Curtiss collection pspA_(EF5668) sequence pBR322 Cloning vector, Ap^(r), Cm^(r) 3 pMEG-249 Suicide vector to generate Salmonella ΔilvG3::TT Megan Health Inc araC P_(BAD) lacl mutant, Ap^(r), Cm^(r) pMEG-443 Suicide vector to generate Salmonella ΔasdA16 26 mutant, Ap^(r), Cm^(r) pMEG-493 Suicide vector to generate Salmonella Δcrp-28 Megan Health Inc mutant, Ap^(r), Cm^(r)

REFERENCES

[0129] Curtiss III, R., S. B. Porter, M. Munson, S. A. Tinge, J. O. Hassan, C. Gentry-Weeks, and S. M. Kelly. 1991. Nonrecombinant and recombinant avirulent Salmonella live vaccines for poultry colonization, p.169-198. In Control of Human Bacterial Enteropathogens in Poultry. Academic Press, Inc., N.Y.

[0130] McDaniel L S, Scott G, Widenhofer K, Carroll J M, Briles D E. 1986. Analysis of a surface protein of Streptococcus pneumoniae recognised by protective monoclonal antibodies. Microbial Pathog. 1:519-31.

[0131] McDaniel, L. S., J. S. Sheffield, E. Swiatlo, J. Yother, M. J. Crain, and D. E. Briles. 1992. Molecular localization of variable and conserved regions of pspA, and identification of additional pspA homologous sequences in Streptococcus pneumoniae. Microbial Pathog. 13:261-269.

[0132] Sampson, J. S., Z. Furlow, A. M. Whitney, D. Williams, R. Facklam, and G. M. Carlone. 1997. Limited diversity of Streptococcus pneumoniae psaA among pneumococcal vaccine serotypes. Infect Immun 65:1967-1971.

Example 1 Construction of Antigen Expression Vector

[0133] General DNA procedures. The plasmids used in the construction of the vectors are listed in Table 1. DNA manipulations were carried out as described in the procedures of Sambrook et al. (43). Transformation of E. coli and Salmonella was done by electroporation (Bio-Rad, Hercules, Calif.). Transformants containing Asd⁺ plasmids were selected on L agar plates without diaminopimelic acid (“DAP”). Only clones containing the recombinant plasmids were able to grow under these conditions. Transfer of recombinant suicide plasmids to Salmonella was accomplished by conjugation using E. coli MGN-617 (Asd⁻) (42) as the plasmid donor. Bacteriophage P22HT int-mediated general transduction was performed by standard methods (50). PCR amplification was employed to obtain DNA fragments for cloning and for verification of chromosomal deletion mutations. The PCR conditions were as follows: denaturation at 95° C. for 20 sec; primer annealing at 55° C. for 20 sec; polymerization at 72° C. for 2 min; and a final extension at 72° C. for 10 min. Nucleotide sequencing reactions were performed using ABI prism fluorescent Big Dye Terminators according to the manufacturer's instructions (PE Biosystems, Norwalk, Conn.).

[0134] Construction of Asd⁺ vectors to use in the antigen expression. The construction of Asd⁺ vectors is also described in PCT/US01/13915, which is herein incorporated by reference. Carrier bacteria strains harboring mulicopy Asd⁺ vectors (pBRori or pUCori) containing the entire asd gene with its promoter synthesized the Asd protein at a much higher level than necessary to complement the chromosomal asd mutation in a balanced-lethal host-vector system. In fact, the 200 to 300 fold excess production of Asd in a strain such as ×8554 (ΔasdAl6) with the pYA3334 Asd⁺ vector (pUCori) increases the generation time slightly and the LD₅₀ ten-fold compared to the same strain with an Asd⁺ vector with the pSC10lori or pl5Aori. In an attempt to reduce the level of Asd, the asd promoter region was deleted to determine whether there would be sufficient transcription to permit a promoterless asd gene to complement the chromosomal ΔasdA16 mutation. The asd gene sequence was amplified by PCR starting at base pair 286 and ending on base pair 1421 of the S. typhimurium asd sequence (GenBank accession number AF015781) with an N-terminal BglII site and a C-terminal XbaI site. This sequence contains the Shine-Dalgarno (SD) sequence for ribosome recognition but lacks the -35 and -10 promoter sequence and ends just after the asd gene TAG stop codon. The Bgll-XbaI DNA fragment was used to construct Asd⁺ vectors pYA3342 (pBR on) and pYA3341 (pUC on) (Table 1). It was possible to clone this fragment onto pSC10lori or pl5Aori vectors but this did not result in sufficient Asd to permit construction of a balanced-lethal host-vector system with strains such as ×8554 which could grow in the absence of DAP. Both pYA3342 and pYA3341 complemented the asd mutations of E. coli ×6212 and S. typhimurium ×4550. Salmonella strains possessing pYA3342 and pYA3341 produced significantly reduced amounts of Asd protein (39 kDa) compared to strains containing plasmids that had asd genes with the asd native promoter (FIG. 1). pYA3342 and pYA3341 in a Δasd S. typhimurium strain such as ×8554 yielded recombinants that had wild-type LD₅₀s following oral inoculation of BALB/c mice. Plasmid pYA3342 was used for further construction (FIG. 2A).

[0135] For the translocation of foreign antigen into the periplasmic space of Salmonella, a recombinant plasmid was constructed by cloning a DNA fragment specifying the signal sequence of β-lactamase. A 105 bp DNA fragment (nucleotides 4049 to 4153 of accession number J01749) of the β-lactamase gene was PCR-amplified from the pBR322 DNA template using a pair of primers, ([N-terminal], 5′GCATTCATGAGTATTCAACATTTCC3′ [SEQ ID NO:4] and ([C-terminal], 5′CCGGAATTCTTCAGCATCTTTTACT3′ [SEQ ID NO:5]). The PCR-amplified fragment included the N-terminus of β-lactamase from the ATG start codon through the signal sequence (23 amino acids) plus 12 amino acids of the N-terminus of the mature β-lactamase (SEQ ID NO:2). These additional 12 amino acid residues were included to increase the efficiency of secretion of the recombinant protein (51). The 105 bp PCR product was digested with BspH1 and EcoRI enzymes and cloned into the Ncol (compatible with the BspHI site) and EcoRI sites of the Asd⁺ vector pYA3342, resulting in plasmid pYA3493 (FIG. 2B). The in-frame position of the β-lactamase signal sequence was confirmed by nucleotide sequencing. Transcription promoted by P_(trc) can be stopped by the 5STIT2 transcriptional terminator located following the multi cloning sites. pYA3493 was stably maintained for 50 or more generation in E. coli ×6212 and S. typhimurium (Δasd) hosts grown in the presence or absence of DAP.

[0136] Construction of the rPspA-expressing plasmid. A highly immunogenic α-helical region of PspA from amino acid residues 3 to 257 (765 bp; 255 amino acids) of the mature PspA_(Rx1) protein (588 amino acids) was selected to use as a test antigen in antigen delivery by a Salmonella carrier. The 765 bp DNA fragment of the pspA gene of S. pneumonia Rx1 was PCR-amplified from the pYA3193 DNA template with a pair of primers ([N-terminal], 5′CCGGMTTCTCTCCCGTAGC-CAGTCAGTCT3′ [SEQ ID NO:8], and the same C-terminal primer used in the construction of histidine [6×]-tagged PspA which introduces the TAA TAG stop codons after the pspA coding sequences [SEQ ID NO:7]). The PCR product, digested with EcoRI and HindIII enzymes, was cloned into EcoRI and HindIII sites of pYA3493, resulting in pYA3494 (FIG. 3). The in-frame fusion of the rPspA with the β-lactamase signal sequence was confirmed by nucleotide sequencing. E. coli ×6212 harboring pYA3494 expressed rPspA as approximately 1% of the total cell protein.

[0137] Purification of recombinant PspA. For overexpression of histidine (6×)-tagged PspA, a fragment of the pspA gene of S. pneumoniae Rx1 was PCR-amplified from pYA3193 (36) template DNA using a pair of primers ([N-terminal] 5′CCGGAATTCATCACCATCACCATCACTCTCCC-GTAGCCAGTCAGT3′ [SEQ ID NO:6], and [C-terminal] 5′GGGAAGCTTCTATTATTCTACA-TTATTGTT3′ [SEQ ID NO:7]). The 0.8 kb amplified fragment was then cloned into the pYA3342 vector, resulting in pYA3496 (Table 1). The N-terminal primer contains an EcoRI site and six consecutive histidine codons (alternate use of CAT and CAC) for histidine (6×) tagging at the N-terminus. The C-terminal primer specifies two consecutive stop codons (TAA TAG) followed by a HindIII site. In-frame cloning was confirmed by nucleotide sequencing. E coli ×6212 harboring pYA3496 expressed a large amount of soluble histidine (6×)-tagged rPspA in its cytoplasmic fraction. According to manufacturer's protocol (Qiagen, Valencia, Calif.), rPspA protein was purified by an affinity purification process with Ni²⁺-nitrilotriacetic acid-agarose support. The protein purity was verified by Coomassie blue staining of SDS-PAGE gels, and the total amount of purified protein was determined by using the Pierce protein assay kit (Pierce, Rockford, Ill.) with BSA as a standard. An immunoblot with the Xi126 PspA monoclonal antibody (31) was performed to confirm the purified protein.

Example 2 Construction of Carrier Bacteria

[0138] Bacterial strains, media and growth conditions. Bacterial strains are listed in Table 1. Bacteriophage P22HTint (45) was used for generalized transduction. Escherichia coli and S. typhimurium cultures were grown at 37° C. in Lennox broth (28) or Luria-Bertani (LB) broth, or on LB agar (1). MacConkey agar (Difco, Detroit, Mich.) supplemented with 1% sugar was used for fermentation assays. The utility of Asd⁺ plasmids in bacterial live vaccines is described elsewhere (35). When required, antibiotics were added to culture media at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 30 μg/ml; kanamycin, 50 μg/ml; tetracycline, 15 μg/ml. Diaminopimelic acid (DAP) was added (50 μg/ml) for the growth of Asd⁻ strains (35). LB agar containing 5% sucrose was used for sacB gene-based counter selection in the allelic exchange experiments (17). S. pneumoniae WU2 was cultured on Brain heart infusion (BHI) agar containing 5% sheep blood or in Todd-Hewitt broth plus 0.5% yeast extract (THY) (6).

[0139] Construction of a S. typhimurium vaccine strain. The Δcrp mutation was introduced into S. typhimurium ×3339 by allelic exchange using the suicide vector pMEG-493 to yield ×8499. The presence of the 680 bp deletion was confirmed by PCR with a primer set flanking crp (5′-AAAG-TCGCAATGGMGGC-3′ [SEQ ID NO:9] and 5′-CGTAGACGACGATGGTCTTG-3′ [SEQ ID NO:10]) and a strain phenotype of Mal⁻ and non motility. The ΔasdA16 mutation was then introduced into ×8499 using P22HTint transduction from ×8554 with the suicide vector pMEG-443 integrated into a strain with the ΔasdA16 mutation followed by sucrose selection to eliminate the suicide vector to yield ×8501 (26). The presence of the 1,242 bp asd deletion in ×8501 was confirmed by PCR using flanking asd primer set (5′-CGGAAATGATTCCCTTCCTAACG-3′ [SEQ ID NO:11] and 5′-TATCTGCGTCGTCCTACCTTCAG-3′ [SEQ ID NO:12]) (26).

[0140] Characterization of phenotype. MacConkey agar supplemented with 1% maltose was used to detect the phenotype of Salmonella crp mutants. Motility was evaluated by observing Salmonella spread on a semisolid medium composed of 1% casein enzyme hydrolysate, 0.5% NaCl, and 0.5% agar. Triphenyltetrazolium chloride (50 μg/ml) was added to motility medium to observe Salmonella as red-colored cells. The presence of the ΔasdA16 mutation in Salmonella was confirmed by inability of the strain to grow on media without DAP (35). Lipopolysaccharide profiles of Salmonella strains were examined by described methods (23).

Example 3 Expression and Secretion of Antigen

[0141] Expression and subcellular localization of rPspA in Salmonella. A S. typhimurium strain was constructed to examine expression and subcellular localization of rPspA. The atrB13::MudJ allele (14), causing constitutive expression of β-galactosidase, in S. typhimurium JF2430 was transduced into S. typhimurium ×8554 by P22HT int-mediated generalized transduction (50), resulting in ×8599 (hisG ΔasdA16 atrB13::MudJ). ×8599 was Lac⁺ on MacConkey agar plus lactose and DAP. β-galactosidase production from the atrB13::MudJ allele in ×8599 was used as a cytoplasmic protein marker and as an indicator of membrane leaking in the examination of subcellular fractionations. To observe rPspA expression, plasmid pYA3494 was introduced into S. typhimurium ×8599. ×8599 harboring pYA3493 (vector alone) was used as the control.

[0142] With the expectation of the periplasmic secretion of the rPspA, various subcellular fractions including cytoplasm, periplasm, outer membrane, and culture supernatant of ×8599 (pYA3494) were prepared to examine the location of rPspA. Although the calculated size of rPspA was approximately 30 kDa, PspA-specific monoclonal antibody Xi126 reacted with an approximately 35 kDa protein (FIG. 4). Aberrant migration of a PspA protein has been seen in previous studies (36, 52). Although a large amount of the rPspA resided in the cytoplasmic fraction, half of the rPspA was detected in the periplasmic fraction and the culture supernatant fluid. Little or no rPspA was detected in the outer membrane fraction. Densitometry analyses of immuno reactive bands showed that approximately 50% of the rPspA was located in both periplasm (25%) and culture supernatants (25%). In the immunoblot analyses of subcellular fractions with anti-β-galactosidase and -OmpC monoclonal antibodies, the β-galactosidase and OmpC proteins were detected in the cytoplasm and outer membrane fractions, respectively, suggesting that the rPspA detected in the periplasmic fraction and culture supernatant fluid was actively secreted instead of resulting from non-specific membrane leaking or cell death.

[0143] Recombinant S. typhimurium Δcrp-28 vaccine expressing rPspA antigen. pYA3493 (vector control) and pYA3494 encoding rPspA were electroporated into the Δcrp-28 ΔasdA16 strain ×8501. S. typhimurium ×8501 (Δcrp-28 ΔasdA16) vaccine strain containing pYA3494 expressed the rPspA protein at an approximate molecular mass of 35 kDa. In the analyses of Coomassie blue-stained SDS-PAGE, the amount of rPspA protein was as much as approximately 1-2% of total proteins of ×8501 (pYA3494) strain (FIG. 5). With results consistent with those seen in the rPspA localization analysis [75% of rPspA cell-associated (50% cytoplasm and 25% periplasm) and 25% of rPspA secreted], the rPspA expressed in the ×8501 vaccine strain was secreted into the culture supernatant along with other secreted proteins. To examine the stability of plasmids pYA3493 and pYA3494 in Salmonella ×8501 in vitro, ×8501 cells containing pYA3493 and pYA3494 were cultured with daily passage for five consecutive days in L broth containing DAP. All ×8501 clones examined (300 clones/day) kept the Asd⁺ plasmid pYA3493 and pYA3494, indicating that pYA3493 and pYA3494 were very stable in the ×8501 vaccine strain. Cells obtained from the last day culture of the stability test expressed similar amounts of the 35 kDa rPspA compared to those from the first day (data not shown), suggesting stable expression of rPspA without rearrangements.

[0144] Methods

[0145] SDS-PAGE and immunoblot analyses. Protein samples were boiled for 5 min and then separated by discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were visualized by Coomassie brilliant blue R250 (Sigma, St. Louis, Mo.) staining. For immunoblotting, proteins separated by SDS-PAGE were transferred eletrophoretically to nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin in 10 mM Tris-0.9% NaCl (pH 7.4) and incubated with mouse monoclonal antibodies specific for PspA (Xi126) (31), OmpC (48), or, β-galactosidase (Sigma), and then with a peroxidase-conjugated goat anti-mouse immunoglobulin G (Bio-Rad). Immuno-reactive bands were detected by the addition of 4-chloro-1-naphthol (Sigma) in the presence of H₂O₂. The reaction was stopped after two minutes by washing with several large volumes of deionized water.

[0146] Salmonella subcellular fractionation. The periplasmic fraction was prepared by a modification of the lysozyme-osmotic shock method (54). Cultures grown in LB broth to 0.8 OD₆₀₀ were centrifuged at 7,000×g for 10 min and the supernatant fluid saved for analysis of secreted proteins. The cell pellets were resuspended in 800 μl of 100 mM Tris-HCl buffer (pH 8.6) containing 500 mM sucrose and 0.5 mM EDTA. Hen egg-white lysozyme (40 μl of 4 mg/ml stock solution) was added, followed immediately by the addition of 3.2 ml of 50 mM Tris-HCl buffer (pH 8.6) containing 250 mM sucrose 0.25 mM EDTA, and 2.5 mM M⁹Cl₂. After gentle agitation, the suspension was incubated for 15 min in an ice bath. Cells were removed by centrifugation at 7,000×g for 6 min followed by filtration of the supernatant through a 0.45 μm filter. The filtered supernatant fluid served as the periplasmic fraction. Cells resuspended in 4 ml of 20 mM Tris-HCl (pH 8.6) were disrupted by two passages through a French pressure cell (American Instrument Company, Silver Spring, Md.). Cell lysates were centrifuged at 7,000×g at 4° C. for 6 min to remove unbroken cells. The supernatant fluid was then centrifuged at 132,000×g at 4° C. for 1 h to separate the soluble fraction and insoluble cell envelopes. The soluble fraction served as the cytoplasmic proteins. To isolate the outer membrane fraction, total envelope pellets were suspended in 4 ml of 20 mM Tris-HCl (pH 8.6) containing 1% sarkosyl and incubated for 30 min on ice. The outer membrane fraction was obtained as a pellet after centrifugation at 132,000×g at 4° C. for 1 h. The pellet was resuspended in 4 ml of mM Tris-HCl buffer (pH 8-6). The original culture supernatant was filtered (0.22 μm filter) and secreted proteins were precipitated with 10% trichloroacetic acid (1 h, 4° C.). An equivalent amount of each fraction sample was separated by SDS-PAGE for western blot analysis. Using the outer membrane protein preparation procedure described above, Salmonella outer membrane proteins (SOMPs) were prepared from S. typhimurium ×4746 cells grown in LB broth without galactose for analysis by ELISA. SOMPs obtained from ×4746 preclude LPS O-antigen contamination.

Example 4 Characterization of Immune Response and Demonstration of Protective Immunity

[0147] Immunization of mice. Two groups of 5 inbred 7-week old female BALB/c mice were deprived of food and water for 4 h before infection. The recombinant S. typhimurium ×8501 (pYA3494) vaccine (1.9×1 09 CFU in 20 μl of phosphate-buffered saline containing 1% gelatin [BSG]) grown in LB broth to 0.8 OD₆₀₀ was orally administered to BALB/c mice. The recombinant S. typhimurium ×8501 (pYA3493) vaccine (2×10⁹ CFU in 20 μl of BSG) was used as a vector control. Food and water were returned to the immunized mice 30 min after immunization. Blood was obtained by retro-orbital puncture with heparinized capillary tubes at biweekly intervals. Following centrifugation at 4,000×g for 5 min, the serum was removed from the whole blood and stored at −20° C. Vaginal secretion specimens were collected in a 50 μl BSG wash and stored at −20° C. (57).

[0148] Pneumococcal challenge. To observe the immune responsiveness of pneumococcal infection after vaccination, a sublethal dose of 3.8×10⁵ S. pneumoniae WU2 CFU in 200 μl of BSG was administered by intravenous (i.v.) injection to S. typhimurium vaccine-immunized BALB/c mice at sixteen weeks after primary immunization. The ability of the Salmonella-PspA vaccine to protect the immunized mice against S. pneumoniae was assessed by intraperitoneal (i.p.) challenge with 4.8×10³ CFU of S. pneumoniae in 100 μl of BSG. The LD₅₀ of S. pneumoniae WU2 in BALB/c mice was >10⁶ by i.v. and <10² by i.p.

[0149] ELISA. ELISA was used to assay antibodies in vaginal secretions and serum to S. typhimurium LPS and SOMPs, and rPspA. Polystyrene 96-well flat-bottom microtiter plates (Dynatech Laboratories Inc., Chantilly, Va.) were coated with S. typhimurium LPS (100 ng/well, Sigma), SOMPs (100 ng/well), or purified rPspA (100 ng/well). Antigens suspended in sodium carbonate-bicarbonate coating buffer (pH 9.6) were coated with 100 μl volumes in each well. The coated plates were incubated at 37° C. for 1 h followed by an overnight incubation at 4° C. Free binding sites were blocked with a blocking buffer (PBS [pH 7.4]-0.1% Tween 20 and 1% bovine serum albumin). Vaginal secretions and sera obtained from the same experimental group (5 mice/group) were pooled and diluted 1:10 and 1:600, respectively. A 100 μl volume of diluted samples was added to individual wells in duplicate and incubated for 2 h at 37° C. Plates were treated with biotinylated goat anti-mouse IgG, IgG1 or IgG2a (Southern Biotechnology Inc., Birmingham, Ala.) for sera and IgA for vaginal secretions. Wells were developed with streptavidin-alkaline phosphatase conjugate (Southern Biotechnology) followed by p-nitrophenylphosphate substrate (Sigma) in diethanolamine buffer (pH 9.8). Color development (optical density) was recorded at 405 nm using an automated ELISA plate (model EL31 1SX; Biotek, Winooski, Vt.). Absorbance readings two times higher than pre-immune serum baselines were considered positive reactions.

[0150] Immune responses in mice by oral immunization with the recombiuant S. typhimurium vaccines. All mice orally administered with a single dose of 1.3×10⁹ CFU S. typhimurium ×8501 (pYA3493, vector control) survived for the 30 day monitoring period. Those mice were protected against wild-type ×3339 (LD₅₀<10⁶) challenge (1.7×10⁹ CFU) 30 days after initial administration. There were no survivors in a group of unimmunized mice challenged with 1.7×10⁷ CFU of ×3339. These results indicate that ×8501 with an Asd⁺ vector is avirulent for mice and elicits a protective immune response against S. typhimurium challenge.

[0151] A single dose of S. typhimurium ×8501 (pYA3494) (1.9×10⁹ CFU) or ×8501 (pYA3493) (control, 2×10⁹ CFU) was orally administered to 7 week-old female BALB/c mice. All immunized mice survived and no signs of disease in the immunized mice during the entire experimental period were observed. The antibody responses to Salmonella LPS and outer membrane proteins (SOMPs), and to the foreign antigen rPspA were measured from the sera and the vaginal secretions of the immunized mice. The serum IgG responses to LPS, SOMPs and rPspA are presented in FIG. 6. At two weeks after administration, little IgG responses to the antigens were observed. Maximal anti-LPS, -SOMPs and -rPspA IgG levels without boost immunization were detected at 6 weeks post immunization, Anti-LPS IgG levels were lower than IgG specific for SOMPs and rPspA.

[0152] At 17 weeks post immunization, mice were infected intravenously with a sublethal dose (3.8×10⁵ CFU) of the virulent S. pneumoniae WU2 strain to monitor the changes of anti-rPspA antibody titers. Sublethal i.v. infection with S. pneumoniae did not kill mice immunized with ×8501 (pYA3493) and ×8501 (pYA3494) vaccines. Because native PspA is a highly immunogenic pneumococcal surface protein, the pneumococcal challenge induced and boosted rPspA specific immune responses in ×8501 (pYA3493) and ×8501 (pYA3494) immunized mice, respectively (FIG. 6). In comparison to the anti-rPspA IgG level (OD₄₀₅ 0.81) at 12 weeks post S. typhimurium ×8501 (pYA3494) immunization, the pneumococcal challenge boosted 53% more anti-rPspA IgG (OD₄₀₅ 1.24) one week later. This suggests that S. typhimurium ×8501-rPspA vaccine induces immunological memory for a rapid responsiveness to subsequently administered PspA antigen. Importantly, the anti-rPspA IgG levels were higher than those to Salmonella LPS and SOMPs. A comparison of maximum IgG levels induced to each antigen by the ×8501 (pYA3494) vaccine reveals that the anti-rPspA IgG level (OD₄₀₅ 1.1) was 36% higher than for LPS (OD₄₀₅ 0.7) and 27% higher than for SOMPs (OD₄₀₅ 0.8). In another experiment in which BALB/c mice were orally immunized with ×8501 (pYA3494), the anti-PspA and anti-LPS IgG titers were A₄₀₅=2.4 and A₄₀₅=1.65, respectively, 8 weeks after immunization; A₄₀₅=2.65 and A₄₀₅=1.33, respectively, 3 weeks after a boost at 16 weeks after primary immunization. Anti-rPspA IgG was not detected in sera obtained from mice immunized with ×8501 (pYA3493), the vector control vaccine. The ×8501 (pYA3493) vaccine elicited anti-LPS and -SOMPs IgG responses with similar kinetics and levels to those induced by ×8501 (pYA3494). These results suggest that Salmonella-delivered rPspA antigen had minimal influence on the immune response to Salmonella itself.

[0153] IgA levels, mostly secretory IgA, for LPS, SOMPs and rPspA were measured from the vaginal fluids of immunized mice. Vaccines ×8501 (pYA3493) and ×8501 (pYA3494) elicited anti-LPS and anti-SOMPs IgA. rPspA specific IgA was detected in the vaginal fluids from mice immunized with ×8501 (pYA3494) but not ×8501 (pYA3493) vector-only control (FIG. 7).

[0154] IgG isotype analyses. The type of immune responses to Salmonella LPS and SOMPs and the rPspA was further examined by measuring the levels of IgG isotype subclasses IgG2a and IgG1. The ThI-helper cells direct cell-mediated immunity and promote class switching to IgG2a, and Th2 cells provide potent ‘help’ for B cell antibody production and promote class switching to IgG1 (38, 49). IgG2a isotype dominant responses were observed for the Salmonella LPS and SOMPs antigens (FIG. 8). The ratios of IgG2a/IgG1 for anti-LPS and -SOMPs in sera obtained from mice immunized with X8501 (pYA3493) were similar to those observed in mice immunized with ×8501 (pYA3494). The ratios of IgG2a/IgG1 for anti-SOMPs (ranging from 6.4 to 11.5) are higher than that for LPS (ranging from 1.1 to 2.5). Th1-type dominant immune responses are frequently observed after attenuated Salmonella immunization (29, 39). In contrast to the type of immune responses to LPS and SOMPs, Thl- and Th2-type mixed responses were observed for the rPspA antigen. Although the IgG2a levels were higher than IgG1 levels in the early phase (up to 8 weeks post immunization), the level of anti-rPspA IgG1 isotype antibodies gradually increased. After 10 weeks post immunization, a 1:1 ratio of IgG2a to IgG1 or IgG1 dominant responses were detected (FIG. 8). The pneumococcal i.v. challenge maintained the IgG1 dominant immune responses to PspA as seen before challenge.

[0155] Evaluation of protective immunity. To examine the ability of Salmonella-rPspA vaccines to protect against pneumococcal infection, BALB/c mice were immunized with either S. typhimurium ×8501 (pYA3493) (1.3×10⁹ CFU dose) or ×8501 (pYA3494) (1.7×10⁹ CFU dose). Ten weeks after initial immunization, a second 10⁹ CFU dose of each vaccine was administered. We did not detect weakness or disease signs in vaccinated mice during the immunization periods. At 5 weeks after the second immunization, mice were challenged intraperitoneally with 4.8×10³ CFU of S. pneumoniae WU2. Sixty percent of the mice immunized with ×8501 (pYA3494) were protected from pneumococcal challenge. This challenge dose killed 100% of unimmunized and ×8501 (pYA3493) immunized mice (Table 2). Following challenge mice unimmunized or immunized with ×8501 (pYA3493) died much quicker with a mean day of death of 2 compared to mice immunized with ×8501 (pYA3494) which had a mean day of death of 5. TABLE 2 Oral immunization of rPspA-expressing S. typhimurium χ8501 (pYA3494) vaccine protects BALB/c mice against challenge with virulent S. pneumoniae WU2 strain RPspA Protection^(c) Vaccines^(a) Expression^(b) (% alive) Days to death P value^(d) χ8501 (pYA3494) + 60 5, 5 >21, >21, >21 χ8501 (pYA3493) − 0 1,2,2,3,3 0.0137 Unimmunized N/A 0 1,2,2,2,3 0.0125

Example 5 Construction of Plasmid pYA3605, Comprising PspA_(EF5668)/PspA_(Rx1) Fusion

[0156] The vast majority of S. pneumoniae serotypes can be grouped into two families (Family 1 and Family 2) with regard to the presence of PspA antigens that share significant immunological relationships as determined by the use of a large bank of monoclonal antibodies against various PspA proteins and the analysis of PspA proteins from a great diversity of S. pneumoniae strains of diverse capsular polysaccharide serotypes. FIG. 9 diagrammatically depicts the familial relationship among many of these diverse serotypes. PspA proteins from Family 1 are not closely related to PspA proteins from Family 2. To construct pYA3605, which comprises polynucleotides encoding fragments of PspA from both Family 1 and Family 2 S. pneumoniae strains, S. pneumoniae strain EF5668 was selected to represent Family 2 and S. pneumoniae strain Rx1 wa selected to represent Family 1. To construct a recombinant attenuated vaccine that would induce an antibody immune response that would be protective against infection with the greatest diversity of S. pneumoniae capsular polysaccharide serotype strains, the coding sequences for the α-helical domains of the PspA antigens from the S. pneumoniae EF5668 and Rx1 strains were fused.

[0157]FIG. 10 gives the nucleotide sequence for the N-terminal portion of the EF5668 pspA gene (SEQ ID NO:13 represents the nucleotide sequence and SEQ ID NO:26 represents the translation product sequence), including the signal sequence and the α-helical domain (as depicted in SEQ ID NO:1). FIG. 11 lists all the oligonucleotide primers to first clone the EF5668 α-helical domain into pYA3493 to yield pYA3594 (FIG. 12) and then proceed with the construction of pYA3605 with the EF5668-Rx1 fusion of the α-helical domains of both PspA antigens (FIG. 12). Primers 1 and 2 (SEQ ID NO:14 and SEQ ID NO:15, respectively) (FIG. 11) were used to amplify by PCR an 831 bp sequence of the EF5668 pspA sequence present on pKSD2106 (McDaniel et al., Infect. Immune. 1998. 66:4748-4754), which encodes amino acids 79 to 353 of the mature PspA protein after cleavage of the 31 amino acids in the PspA signal sequence. Primer 2 (SEQ ID NO:15; FIG. 11) also specified the stop codons TGA TM after PspA_(EF5668) amino acid 353. The PCR product was digested with EcoRI and HindIII and ligated to the Asd⁺ bla SS vector pYA3493, which also had been digested with EcoRI and HindIII.

[0158] This new plasmid was transformed into ×6212(pYA232) and designated pYA3594 (FIG. 12). To facilitate construction of the fusion PspA protein, Primers 3 and 4 (SEQ ID NO:16 and SEQ ID NO:17, respectively; FIG. 11) were used for inverse PCR with pYA3594 (FIG. 12) to introduce a PstI recognition site between amino acids 308 and 309 in the mature PspA_(EF5668) sequence (SEQ ID NO:26; FIG. 10). The DNA fragment generated by inverse PCR was digested with PstI and ligated to yield pYA3604 (FIG. 12), which was transformed into strain ×6212 (pYA232). DNA primers 5 and 6 (SEQ ID NO:18 and SEQ ID NO:19, respectively) were then used to amplify by PCR a 777 bp DNA sequence from pYA3494 (FIG. 12) encoding amino acids 3 to 257 of PspA_(RX1) and the two stop codons TM TAG. This DNA fragment was digested with PstI and HindIII and ligated with pYA3604, which was also digested with PstI and HindIII, to generate pYA3605 (FIG. 12), which was transformed into ×6212 (pYA232). This construction led to the deletion of 45 amino acids from the Psp_(AEF5668) protein encoded in pYA3594.

[0159] The final construct thus specifies the β-lactamase signal sequence and first 12 amino acids, two codons for the EcoRI site, 230 amino acids of the PspAEF5668 protein (mature amino acids 79 to 308), two codons for the PstI site and 255 amino acids of the PspA_(Rx1) protein (mature amino acids 3 to 257) followed by two stop codons (SEQ ID NO:25; FIG. 13). The entire fusion encoded in pYA3605 has 524 amino acids with 501 remaining after cleavage of the signal sequence.

[0160] All of the recombinant plasmids described above were transformed into ×6212 (pYA232). pYA232 has a pSC101 replicon that is compatible with the pBR ori present in all the plasmid constructs described above. pYA232 also possesses the lacl^(q) gene so the expression of genes under the control of the P_(trc) promoter is repressed. This requires the addition of the inducer IPTG to relieve Lacl^(q) repression and permit transcription of the PspA encoding genes in all of the recombinant vectors described above. Growth of ×6212 (pYA232, pYA3605) in Luria broth containing 1 mM IPTG and analysis of the proteins synthesized by SDS-PAGE followed by western blot analysis with MAb Xi126 against the Rx1 PspA and MAb XiR278 against the EF5668 PspA (FIG. 14) revealed a fusion protein of the anticipated size and possessing determinants recognized by both monoclonal antibodies.

Example 6 Construction of pYA3620 Containing the N-Terminal Signal Sequence and C-Terminal Portions of the β-Lactamase Gene

[0161] In addition to the 12 amino acids at the N-terminal end of the mature β-lactamase protein being important for β-lactamase secretion, some evidence suggests that the 21 amino acid sequence from the C-terminal end of β-lactamase (SEQ ID NO:3) might also enhance secretion across the cytoplasmic membrane. Therefore the Asd⁺ β-lactamase secretion vector pYA3493 (FIG. 15) has been modified to encode the C-terminal 22 amino acids of the β-lactamase protein.

[0162] Oligonucleotide primers 7 and 8 (SEQ ID NO:21 and SEQ ID NO:22, respectively; FIG. 15) were used to amplify by PCR a 660 bp DNA sequence from the β-lactamase gene present in pBR322 (FIG. 15) that specifies ampicillin resistance. This sequence contains the entire C-terminal portion of β-lactamase and because of the restriction enzyme recognition sequences encoded in primers 7 and 8, contains SalI and HindIII restriction sites. This PCR-generated DNA fragment was therefore digested with SalI and HindIII and cloned into pYA3493, which had also been digested with SalI and HindIII. This resulted in the recombinant plasmid pYA3616 (FIG. 15). Using oligonucleotide primers 9 and 10 (SEQ ID NO:23 and SEQ ID NO:24, respectively; FIG. 15) for an inverse PCR reaction with pYA3616 yielded a ˜2.0 kb DNA fragment, which deleted 596 bp of the C-terminal portion of the bla gene initially amplified by PCR, as described above. This DNA fragment was digested with PstI and XbaI to yield a ˜1.8 kb DNA fragment that was ligated to a ˜1.4 kb DNA fragment generated from pYA3493 by PstI and XbaI digestion. The ligated plasmid, which is designated pYA3620 (FIG. 15), was transformed into ×6212. pYA3620 possesses the β-lactamase signal sequence and N-terminal 12 amino acid sequence present in pYA3493 and a 22 amino acid C-terminal amino acid sequence of β-lactamase following a PstI restriction site in pYA3620.

[0163] As constructed, pYA3620 will lead to the synthesis of the β-lactamase signal sequence followed by codons for the first 12 amino acids in the secreted form of β-lactamase and then 24 amino acids encoded in the multiple cloning site and the beginning of the out-of-frame coding sequence for the C-terminal β-lactamase sequence. The TAG stop codon for this peptide is identified by *** in FIG. 16 (SEQ ID NO:25) which presents the nucleotide sequence encoding P_(trc), the β-lactamase signal sequence plus N-terminal β-lactamase 12 amino acids, the multiple restriction enzyme recognition sequences as multiple cloning sites and the β-lactamase C-terminal 21 amino acid sequence present in pYA3620.

[0164] Although the β-lactamase C-terminal sequence is out of frame, the cloning of any sequence encoding a protective antigen can be generated by PCR with N-terminal EcoRI and C-terminal PstI cleavage sites such that an in-frame fusion of the N-terminal β-lactamase sequence to the protective antigen to the C-terminal β-lactamase sequence will result. Insertion of the pspA encoding sequence from pYA3494 into pYA3620 can be achieved by PCR amplification of the pspA DNA fragment in pYA3494 using oligonucleotide primer 5′ CCG GAA TTC TCT CCC GTA GCC AGT CAG TCT (SEQ ID NO:8) to encode an EcoRI site and the N-terminal PspA amino acids 3 to 9 and the primer 5′ AAC TGC AG TTC TAC ATT ATT GTT TTC TTC AGC (SEQ ID NO:27), which encodes the C-terminal PspA amino acids 250 to 257 followed by a PstI site. Digestion of the PCR product with EcoRI and PstI and cloning into pYA3620 similarly digested with EcoRI and PstI will yield the desired in-frame fusion. A similar strategy can be used to clone the sequence encoding the PspA_(EF5668)-PSpA_(Rx1) fusion from pYA3605 into pYA3620.

Example 7 Superior Immune Response to PspA Antigen when Secreted Rather than Retained in Cytoplasm of Recombinant Attenuated Vaccine Strain

[0165] To raise antibodies to purified PspA protein, plasmid pYA3496, which encodes six His residues by alternate CAT and CAC codons preceded by the codons for Met, Gly and lie at the N-terminal end of the PspA protein (amino acids 3 to 257), was constructed. This enabled affinity purification using Ni²⁺-nitrilotriacetic acid-agarose and yielded purified PspA protein specifically recognized by the MAb Xi126. All of the His-PspA protein was retained within the cytoplasm of either E. coli K-12 stain ×6212 or the S. typhimurium UK-1 ×8501 vaccine strain and required lysis of the strain by sonication or by use of a French press for recovery. When ×8501 (pYA3496) was comparatively evaluated for immunogenicity to ×8501 (pYA3494), both strains induced an immune response to the LPS antigen; whereas, the immune response to PspA was substantial following immunization of BALB/c mice with ×8501 (pYA3494) and miniscule after immunization with ×8501 (pYA3496) (FIG. 17).

Example 8 Optimizing Antigen Gene Expression by Replacing Codons Infrequently Used by Salmonella for Highly Expressed Genes with Codons Preferentially Used by Salmonella for Highly Expressed Genes

[0166] Codons for the amino acids leucine, arginine, isoleucine, glycine and proline used in genes of Streptococcus pneumoniae are often codons that strains of Escherichia coli and Salmonella infrequently do not include, especially, in genes that are highly expressed. For this reason, improved expression of genes encoding protective antigens, especially from gram-positive bacteria, can be achieved by eliminating (i.e., changing) any codons within genes encoding, for example, streptococcal protective antigens such as PspA and substituting in their place, codons for the same amino acids that are frequently used in Salmonella and E. coli highly expressed genes (Henaut and Danchin, In Escherichia coli and Salmonella Cellular and Molecular Biology, Vol. 2, 1996, Neidhardt et al., eds., ASM Press, Washington D.C., pp. 2047-2066; Gouy and Gautier, 1982, Nucleic Acids. Res. 10:7055-7074). In this way, expression of the protective antigen gene can be very much enhanced without altering the amino acid sequence of the antigen or the immune response induced to it. We have therefore modified the α-helical domain specifying 255 amino acids representing amino acids 3 to 257 of the mature Streptococcus pneumoniae Rx1 pspA gene as listed in FIG. 18 by replacing nine codons with codons for the same amino acids that are preferentially included in highly expressed genes in Salmonella. We employed a sequential codon replacement strategy using site-directed mutagenesis and PCR methodology for each specific codon or, in some cases, neighboring codon modifications at the same time with reassembly of the intact coding sequence with preferred codons for all amino acids. The nucleotide and amino acid sequences of the modified PspA Rx1 gene and gene product are presented in FIG. 19 with the modified codons indicated in boldface type. The entire DNA sequence was determined by DNA sequencing techniques and there were two base pair changes (underlined in FIG. 19) at the third position of two codons that continued to specify a codon from highly expressed genes for the same amino acid specified by the native PspA Rx1 sequence.

[0167] As indicated in Example 5, there are two major families of S. pneumoniae strains with regard to the immunogenic properties of the PspA protein. Strain Rx1 is in family 1 and we have selected S. pneumoniae strain EF5688 for the cloning of the α-helical domain as a representative of family 2. FIG. 20 provides the nucleotide and amino acid sequences of the modified PspA EF5688 gene and 229 amino acid gene product with the eight modified codons used in Salmonella highly expressed genes indicated in bold-face type. The α-helical domain of the EF5688 PspA protein represents amino acids 79 to 307 in the mature PspA protein (see FIG. 10). To facilitate presentation of the α-helical domains of both PspA proteins by the same vaccine strain, we constructed a fusion of the modified EF5688 (FIG. 20) and Rx1 (FIG. 19) sequences containing improved codons in the same way we used to make the fusion construct present in pYA3605 (FIG. 12) and whose nucleotide sequence is given in FIG. 13. The nucleotide and amino acid sequences of this modified fusion gene product with modified codons indicated in bold-face to optimize expression in Salmonella are presented in FIG. 21. A ctg cag sequence specifying a PstI restriction enzyme cleavage site was inserted by use of specified oligonucleotide primers between the C-terminal end of the PspA_(EF5688) sequence and the N-terminal end of the PspA_(Rx1) sequence (FIG. 21) to enable construction of the fusions. FIG. 22 lists the sequences in each of the oligonucleotide primers used to optimize codons (FIGS. 19 and 20), to construct the fusion and to construct recombinant vectors (see below and FIG. 23).

Example 9 Construction of Expression Plasmid Vectors With EF5688, Rx1 and EF5688-Rx1 Fusion α-Helical PspA Sequences Optimized for Expression due to Replacement of Codons Inefficiently Utilized in Highly Expressed Genes of Salmonella With Codons Preferentially Used by Salmonella for Expression of Highly Expressed Genes

[0168] During the course of constructing the codon-optimized pspA gene sequences as described in Example 8, it was discovered that there was a single base pair mutation in the Rx1 α-helical pspA sequence cloned in pYA3494 (Example 1 and FIG. 3), to result in a mutation that altered the C-terminal 22 amino acids. This was corrected (FIG. 23) in constructing pYA3634 using pYA3494 and the oligonucleotide sequence 2 (FIG. 22) with the corrected pspA Rx1 sequence as a replacement for pYA3494 (FIG. 3). To enable comparative studies on growth, genetic stability, physiological attributes, ability to colonize lymphoid tissues in vivo and to induce immune responses, we have constructed a set of six recombinant plasmids, three with the original α-helical pspA nucleotide sequences as found in S. pneumoniae strains Rx1 and EF5688 separately and as an EF5688-Rx1 fusion and three with the codon optimized improved sequences for pSPARx1 (FIG. 19), pSPA_(EF5688) (FIG. 20) and the pSpA_(EF5688-Rx1) fusion (FIG. 21). The construction of pYA3605 with the native pSPA_(EF5688-Rx1) fusion was diagramed in FIG. 12 with its complete nucleotide and amino acid sequence presented in FIG. 13 along with the N-terminal β-lactamase signal sequence. The construction of the new plasmid pYA3634 with the β-lactamase signal sequence fused to the native pspA_(Rx1) sequence (FIG. 19) is diagramed at the bottom of FIG. 23. The construction of each of the other four plasmids is diagramed in FIG. 23 with an indication of the vectors and oligonucleotide sequences (given in FIG. 22) used. These include pYA3623 with the native PSPA_(EF5688) sequence (FIG. 10), pYA3633 with the codon optimized improved PSPA_(EF5688) sequence (FIG. 20), pYA3635 with the codon optimized improved pSPA_(Rx1) sequence (FIG. 19) and pYA3636 with the codon optimized improved PspA_(EF5688-Rx1) fusion (FIG. 21) all fused to the β-lactamase signal sequence as contained in the original pYA3493 (FIG. 2).

[0169] The resulting six expressions plasmids pYA3634, pYA3623, pYA3605, pYA3635, pYA3633 and pYA3636 are diagramed in FIG. 24. All six plasmids were electroporated first into the E. coli K-12 cloning host ×6212 (Table 1) with pYA232, a pSC101 plasmid with lacI gene, and also into the Δcrp Δasd S. typhimurium vaccine strain ×8501 (Table 1) with selection for Asd⁺ recombinants. These strains were fully characterized with regard to phenotypic and genotypic properties (see Example 2) and the presence of recombinant plasmids of the correct size, containing the intended pspA gene sequence (using PCR and DNA sequence analyses) and specifying synthesis of PspA proteins that would react in western blot analyses with antibodies specific for either or both the Rx1 and EF5688 PspA proteins. PspA synthesis in the ×6212 (pYA232) host required addition of the inducer IPTG to reverse repression of the P_(trc) promoter by the pYA232 specified Lacl repressor. These recombinant strains, listed Table 1, were catalogued, stocked and frozen at −70° C. for long-term storage.

[0170] As described in Example 6, we constructed the Asd⁺ vector pYA3620 (FIG. 16) that possesses both the N-terminal signal sequence and the C-terminal end of the β-lactamase gene to investigate potential improved expression, secretion and/or stability of vaccine strains specifying synthesis of PspA proteins. As noted in the Example below, vaccine strains with the codon optimized improved PSPA_(EF5688) sequence (with or without fusion to the pspA_(Rx1) sequence) grew more slowly and exhibited some instability compared to vaccine strains expressing the native pneumococcal pspA_(EF5688) sequences. We have therefore constructed pYA3637 (FIG. 25) that contains the codon optimized improved pspA_(EF5688) sequence in the pYA3620 vector to evaluate potential improvements in expression, secretion and/or stability compared to a vaccine strain using the vector pYA3633 (FIGS. 23, 24) lacking the fused C-terminal β-lactamase peptide. FIG. 26 provides the entire nucleotide and amino acid sequences for the β-lactamase-signal sequence-improved pspA_(EF5688)-β-lactamase C-terminus fusion as present in pYA3637.

Example 10 Comparative Characterization of Recombinant Attenuated S. Typhimurium Vaccine Strains Specifying PspA Fusion and Non-Fusion Proteins Specified by Non-Codon Optimized and Codon Optimized DNA Sequences

[0171]FIG. 27 presents results of a polyacrylamide gel stained with Coomassie brilliant blue after electrophoresis of proteins from the ×8501 parent and ×8501 with each of the six plasmids. The results clearly demonstrate synthesis of PspA proteins of the expected sizes and in every case reveal that the ×8501 strains with plasmids pYA3633, pYA3636 and pYA3635 with the codon optimized improved sequences for synthesis of the PspA_(EF5688), PspA_(EF5688-Rx1) fusion and PspA_(Rx1) proteins overproduced these proteins in comparison with the ×8501 strains with the plasmids encoding PspA proteins specified by the native pneumococcal nucleotide sequences. All six strains were then evaluated, in comparison to ×8501 (pYA3493) as the vector control, for plasmid stability by growth in LB broth containing DAP with 1 to 1000 dilutions of cultures each day for five days. After 50 generations of growth, cultures were diluted and plated on LB agar containing DAP and replica plating was used to determine the frequency of cells that still possessed the Asd⁺ vector. For all six constructs specifying PspA proteins, some 10 isolates were grown in LB broth in the presence of DAP and evaluated for the synthesis of PspA proteins of the expected molecular size and reacting with antibodies to either or both the Rx1 or EF5688 PspA protein antigens. Five of seven strains retained the Asd⁺ vectors during 50 generations of growth under permissive conditions in the presence of DAP. The exceptions were ×8501 (pYA3633) and ×8501 (pYA3636) where there was significant loss of Asd⁺ vectors on day 3 and thereafter. Similarly, most isolates tested continued to synthesize PspA proteins of the correct size with the exception of the ×8501 (pYA3633) and ×8501 (pYA3636) strains both specifying codon optimized PspA proteins. In accord with these observations, it was determined that ×8501 (pYA3633) and especially ×8501 (pYA3636) grew significantly more slowly than either ×8501 (pYA3623) or ×8501 (pYA3605) with the original pneumococcal non-codon optimized sequences. This result is most likely due to the fact that the strains possessing the plasmids pYA3633 and pYA3636 with codon optimized pspA gene sequences produced much more PspA protein than did ×8501 strains with pYA3634 and pYA3623 having the non-codon optimized sequences as originally cloned from S. pneumoniae strains Rx1 and EF5688, respectively (see FIG. 27 for this evidence). This difference in growth rate was largest in comparisons between ×8501 (pYA3605) with the non-codon optimized fusion sequence and X8501 (pYA3636) that has the codon optimized DNA sequence encoding the PspA EF5688-Rx1 fusion protein. In this case, growth of the latter strain was at half of the rate of the former strain. Interestingly, the Rx1 encoded PspA protein, whether specified by the non-codon optimized sequence (pYA3634) or the codon optimized sequence (pYA3635), was well tolerated by the Salmonella vaccine strain ×8501 with complete stability in the maintenance of Asd⁺ vectors and the synthesis of PspA proteins (see FIG. 27).

[0172] In past studies with recombinant attenuated Salmonella antigen delivery vaccines, it has been observed that immune responses are nearly proportional to the amount of antigen production by the recombinant attenuated Salmonella vaccine. In other words, more antigens produced by the vaccine strain induces higher levels of immune responses. This relationship, however, breaks down if the level of antigen synthesis significantly inhibits the rate of growth and multiplication of the vaccine strain since, in this case, the recombinant attenuated Salmonella vaccine has reduced capacity to invade and colonize internal lymphoid tissues that constitute sites in which immune responses are induced.

Example 11 Construction of Vaccine Strains Expressing Protective Bacterial Antigens to Optimize Colonization of Lymphoid Tissues In Vivo and Induction of High Level Immune Responses

[0173] There are at least two possible solutions to the problem of vaccine strain instability as described in Example 10. In one case, it is possible to re-clone the codon optimized sequences using PCR methods and the oligonucleotide primers listed in FIG. 22 into the Asd⁺ vector pYA3332 (FIG. 28) that possesses the p15A ori specifying a lower plasmid copy number per bacterial cell than specified by the plasmid derivatives used above that were derived from pYA3342 (FIG. 2) that possesses the pBR ori. It should be noted, that pYA3332 and pYA3342 possess nearly identical structures except for the origin of replication. They have the same promoter, multiple cloning site and transcription terminator sequences as indicated in FIG. 2. The introduction of these pYA3332 PspA-specifying plasmids into ×8501 would be expected to result in strains that would grow more rapidly, and more stably maintain the Asd⁺ plasmids but synthesize a somewhat reduced amount of the PspA proteins specified by the codon optimized sequences. This is because the amount of gene product synthesized is proportional to the gene copy number and the fact that the gene copy number is significantly lower on vectors with p15A ori than on vectors with the pBR ori. The level of PspA synthesis would therefore be expected to be about the same as in ×8501 strains possessing Asd⁺ vectors with non-codon optimized pspA sequences encoding the PspA antigen synthesized. These recombinant attenuated vaccine strains would be fully characterized phenotypically and genotypically for genetic stability, growth rate, expression of PspA antigen, colonization of lymphoid tissues and induction of immune responses, all in comparison with recombinant attenuated Salmonella vaccine strains containing recombinant Asd⁺ vectors with the pBR ori. Although this approach would likely yield recombinant attenuated vaccine strains that would colonize lymphoid tissues and induce immune responses that might be superior to those induced by ×8501 (pYA3494) (see Example 4), there is an even better way to achieve high-level colonization of lymphoid tissues and induction of superior immune responses.

[0174] All of the Asd⁺ recombinant vectors cause expression via transcription from the P_(trc) promoter that can be completely repressed to block transcription by the presence of the Lacl repressor protein. This was proven to be so when each of the Asd⁺ plasmids was introduced into ×6212 (pyA232) and PspA synthesis shown to be dependent on addition of IPTG to cause depression of transcription from P_(trc) (Example 9). What is desired, is a recombinant attenuated vaccine strain that would not synthesize PspA antigen due to non-transcription from P_(trc) during growth in vitro and in the initial stages following immunization when the vaccine strain is invading into the GALT and commencing to colonize internal lymphoid tissues after which synthesis of PspA antigen is desired to induce immune responses. To facilitate delayed expression of DNA sequences encoded on Asd⁺ vectors downstream from P_(trc), we have employed the araCP_(BAD) activator-promoter system (Guzman et al., 1995, J. Bacteriol. 177:4121-4130). In this system, a promoterless gene encoding the Lacl protein is fused to the P_(BAD) promoter, so that transcription is dependent on the presence of arabinose that activates the araC gene product to positively cause transcription from P_(BAD). Interestingly, there is essentially no free arabinose in animal tissues such that following colonization of lymphoid tissues transcription of the lad gene ceases with the Lacl repressor decreasing in concentration by half after every cell division to gradually lead to derepression in the expression of gene sequences under the control of P_(trc) on the Asd⁺ plasmid vectors. The araCP_(BAD) lacl construction has therefore been inserted into the ilvG gene within the bacterial chromosome. The ΔilvG3:: TT araC P_(BAD) lad construction present in ×8623 (Table 1) is diagramed in FIG. 29 and the suicide vector for its introduction into the bacterial chromosome of vaccine strains such as ×8501 by methods described by Kang et al. (2002, J. Bacteriol. 184:307-312) is diagramed in FIG. 30. The delayed synthesis of protective antigens in vaccine strains with the ΔilvG3:: TT araC P_(BAD) lacl construction can be extended if the vaccine strain is unable to catabolically break down arabinose (Guzman et al., 1995, 177:4121-4130). This can be accomplished by the introduction of the ΔaraBAD23 mutation (FIG. 29) present in ×8767 (Table 1) using the suicide vector diagramed in FIG. 30. Further delay can be achieved by introducing the ΔaraE25 mutation present in ×8477 (Table 1) that both reduces the rate of uptake and the rate of loss of arabinose from the cytoplasm of vaccine cells. The ΔaraE25 construction is also diagramed in FIG. 29 and the suicide vector for its introduction into the chromosome of vaccine strains is also diagramed in FIG. 30. The methods for generating defined deletion mutations and their introduction into the chromosome using suicide vector technologies was described in Example 2 and more extensively in the manuscript by Kang et al (2002, J. Bacteriol. 184:307-312).

[0175] Following construction of a ×8501 derivative with the ΔilvG3:: TT araC P_(BAD) lacl, ΔaraBAD23 and araE25 mutations, recombinant Asd⁺ plasmid vectors such as pYA3633, pYA3635 and pYA3636 with codon optimized sequences encoding PspA antigens can be introduced by electroporation. These recombinant attenuated vaccine strains would be fully characterized phenotypically and genotypically for genetic stability, growth rate, non-expression of PspA antigen when the strain is grown in the presence of arabinose and gradual ability to synthesize PspA antigen as a consequence of cell division after removal of arabinose from the growth medium, colonization of lymphoid tissues and induction of immune responses. Appropriate control strains would be included for comparison.

Example 12 Construction of Vaccine Strains Specifying Synthesis of the α-Helical Domain of the Streptococcus Pneumoniae PsaA Protein to be Secreted, in Part, by Recombinant Attenuated Salmonella Vaccines to Elicit Superior Immune Responses Against S. Pneumoniae Infection in Immunized Individuals

[0176] In terms of an effective vaccine to prevent infection of humans with diverse Streptococcus pneumoniae serotypes, the synthesis of the α-helical domains of the EF5688 and Rx1 PspA proteins is likely to induce significant protective immune responses. On the other hand, the inclusion of an additional antigen to be synthesized by the vaccine strain would further enhance vaccine efficacy. The PsaA antigen, encoded in the genome of S. pneumoniae 6B, a most prevalent pneumococcal serotype (Butler et al., 1995, J. Infect. Dis. 171:885-889; Jorgenson et al., 1991, J. Infect. Dis. 163:644-646), is a lipidated 32.5 kDa antigen with an N-terminal Cys residue generated after cleavage of the 19 amino acid signal sequence (De et al., 2000, Vaccine 18:1811-1821). The surface PsaA protein is an adhesin used by pneumococci to colonize in the nasal pharynx. Its synthesis is essential for S. pneumoniae virulence (Berry and Paton, 1996, Infect. Immun. 64:5255-5262) and this protein antigen can induce protective immunity to pneumococcus challenge (Briles et al., 2000, Infect. Immun. 68:796-800; Briles et al., 2000, Vaccine 18:1707-1711; Talkington, 1996, Microb. Pathog. 21:17-22). Because of these properties and functions, immunity to PsaA is more effective in blocking colonization in the nasal pharynx and immunity to PspA more effective in blocking systemic infection (Briles et al., 2000, Infect. Immun. 68:796-800). An additional beneficial feature of including the PsaA antigen for expression by a recombinant attenuated Salmonella vaccine is the fact that the amino acid sequence of this protein seems to be almost invariant in some eight psaA genes sequenced from diverse pneumococcus strains to date (analyzed using GenBank deposited sequences). Structural analysis of the PsaA protein causes us to express an α-helical hydrophilic segment commencing with either amino acid 20 (Cys) or 21 (Ala) and ending with amino acid 210 as diagramed in FIG. 31. All four non optimal codons for expression in Salmonella would be optimized for high-level expression in Salmonella by site directed mutagenesis and PCR methodologies and the codon optimized sequence encoding the α-helical domain of PsaA cloned, using suitable oligonucleotide primers, into either pYA3493 (FIG. 2) and/or pYA3620 (FIGS. 15 and 16). These plasmid constructs would be introduced into the ×8501 derivative with ΔilvG3:: TT araC P_(BAD) lacl, ΔaraBAD23 and ΔaraE25 mutations. These constructs would be fully evaluated for phenotype, genotype, stability of plasmid vector, rate of growth, inability to synthesize the PsaA protein when grown in the presence of arabinose and gradual ability following cell division in the absence of arabinose to commence PsaA synthesis, to colonize lymphoid tissues after oral immunization of mice and to induce significant immune responses including protective immunity to challenge with virulent S. pneumoniae strains. All of these procedures have been described in proceeding examples. Upon completion of these evaluations, a decision would be made whether to specify the codon optimized PspA EF5688-Rx1 fusion and the codon optimized PsaA sequence as a fusion in a single vector in the vaccine strains or by two separate vaccine strains that would be used as a vaccine mixture.

Example 13 Construction of Vaccine Strains Specifying Synthesis of Surface Protein Antigens of Various Streptococcus Species and From Other Gram-Positive Bacterial Pathogens to Enable Secretion, in Part, of the α-Helical Domains of these Proteins by Recombinant Attenuated Salmonella Vaccines to Elicit Superior Immune Responses in Immunized Individuals

[0177] There is great similarity in the structural properties of surface protein antigens synthesized by a diversity of Streptococcus species. These surface protein antigens may serve as adhesins to host cell surfaces, interact with extracellular matrix proteins, exhibit antiphagocytic activity, bind choline, and presumably provide additional functions ( ). These proteins all possess highly immunogenic α-helical domains near their N-terminus following cleavage of the signal sequence ( ). These gene products include the choline-binding proteins found on strains of S. pneumoniae, the M proteins on the surface of Group A S. pyogenes strains, surface proteins on the surface of Group B streptococci, the M protein on the surface of S. equi that is responsible for much of the pathology associated with the disease strangles of horses, the antigen I/II of S. mutans and the SpaA antigen of S. sobrinus. In these regards, we have had extensive experience in inducing immune responses using recombinant attenuated Salmonella to the SpaA protein of S. sobrinus (Holt et al., 1982, Infect. Immun. 38:147-156; Curtiss, 1986, J. Dent Res. 65:1034-1045) and the M protein of S. equi (Galán et al., 1989, In D. G. Powell (ed.), Equine Infectious Diseases V, Proceedings of the Fifth International Congress, The University Press of Kentucky, Lexington, Ky.). Others have used attenuated Salmonella to induce immune responses to surface antigens from S. pyogenes (Poirier et al., 1988. J. Exp. Med. 168:25-32) and S. mutans (Huang et al., 2000, Infect. Immun. 68:1549-1556).

[0178] Previous difficulties in constructing stable immunogenetic recombinant attenuated Salmonella vaccines expressing surface antigens of gram-positive bacterial pathogens could be due, in part, to the inability of the gram-negative Salmonella antigen delivery vector to efficiently synthesize and export gram-positive bacterial protein surface antigens in the absence of any instability and toxicity to the vaccine construct. In this regard, the gram-positive signal sequence either does not interact efficiently with or is not processed efficiently by the protein secretion system in gram-negative E. coli and S. typhimurium strains (Nayak et al., 1997, Infect. Immun. 66:3744-3751). The deletion of the signal sequence for the exported gram-positive protein and its replacement with a highly expressed and very efficient signal sequence whose function is optimal in gram-negative enteric bacteria can therefore be predicted to substantially improve the stability and the immunogenetic efficacy of these constructed vaccines as has been demonstrated for the α-helical domain of the S. pneumoniae PspA protein (see Example 4). Thus the use of plasmid vectors such as pYA3493 (FIG. 2) and pYA3620 (FIGS. 15 and 16) to clone codon optimized DNA sequences encoding α-helical domains of surface protein antigens from gram-positive bacterial pathogens such as the species of Streptococcus listed above, Erysipelothrix rhusiopathiae, Bacillus anthracis, Corynebacterium diphtheriae, Clostridium species, etc. for expression and delivery by recombinant attenuated Salmonella vaccine strains would likely generate vaccines that would be efficacious due to their stability and ability to synthesize and deliver the recombinant vector-encoded protective antigen, all contributing to high immunogenicity.

Example 14 Construction of Recombinant Attenuated S. Paratyphi A and S. Typhi Vaccines to Induce Protective Immunity to Humans to Prevent Infections Caused by Streptococcus Pneumoniae, other Streptococcus Species and Other Gram-Positive Bacterial Pathogens

[0179] Evaluation and validation of recombinant attenuated Salmonella vaccines for delivery of antigens specified by DNA sequences encoding protective antigens from other pathogens invariably makes use of strains of S. typhimurium and immunization studies in mice. This is because S. typhimurium induces a disease in mice that is similar to the enteric fever and typhoid fever diseases caused by S. paratyphi A and S. typhi in humans. Unfortunately, S. paratyphi A and S. typhi are human host specific, are unable to infect mice and are rapidly cleared as though they were innocuous strains of bacteria. Since protecting mice against pneumococcal disease does not constitute a worthwhile long-term objective, we have constructed a diversity of S. paratyphi A and S. typhi attenuated strains that can be used as antigen delivery vectors for recombinant antigens that were initially presented to mice by attenuated strains of S. typhimurium to evaluate safety and efficacy. To optimize immunogenicity, these attenuated S. paratyphi A and S. typhi strains possess an RpoS⁺ phenotype (Nickerson and Curtiss, 1997, Infect. Immun. 65:1814-1823; U.S. Pat. Nos. 6,024,961 and 6,383,496). Many of the genetic means to fully attenuate S. typhimurium for mice and to render them highly immunogenic are inadequately attenuating in S. typhi administered to human volunteers. For this reason, we have been developing improved means to attenuate S. paratyphi A and S. typhi to use as antigen delivery vectors for humans. Although some of the S. paratyphi A and S. typhi strains with mutations eliminating functions of the phoPQ operon that might be suitable and safe as vaccine vectors for humans are listed in Table 1, other improved attenuation strategies that we will use for S. paratyphi A and S. typhi are included in a simultaneously submitted patent application entitled “Regulated attenuation of live vaccines to enhance cross protective immunogenicity”. All of these attenuated S. paratyphi A and S. typhi antigen delivery vectors for humans also possess the ΔasdA16 mutation (see Table 1) to enable immediate use of the constructed recombinant Asd⁺ vectors evaluated in S. typhimurium in mice using the standard balanced-lethal host-vector system (U.S. Pat. No. 5,672,345 and pending U.S. patent application entitled “Functional balanced-lethal host-vector systems”) to ensure long-term stability of the recombinant constructs with antigen synthesis in vivo in the absence of any exogenously applied selective pressure. Importantly, as a safety feature, all such recombinant attenuated Salmonella vaccines are completely sensitive to all antibiotics such that an individual experiencing an unexpected adverse consequence of immunization could be effectively treated with antimicrobial agents. This benefit does not exist if a live attenuated bacterial vaccine possesses genes for antibiotic resistance in its chromosome or more often on plasmid vectors specifying recombinant antigens. For this reason, regulatory agencies charged with evaluation of vaccines for safety and efficacy will almost always disallow presence of antibiotic resistance genes, especially if those antibiotics are potentially useful in treating infections by unattenuated bacteria of the same type as used as the vaccine host component. This benefit is generally precluded in using live recombinant attenuated viral vaccine vectors since there are few effective antiviral drugs available.

1 72 1 275 PRT Streptococcus pneumoniae 1 Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp Lys Lys Ile 1 5 10 15 Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala Phe Ala Thr 20 25 30 Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser Glu Leu Ala Glu Thr 35 40 45 Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala Glu Val Ala Lys Lys 50 55 60 Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val Glu Lys Asn Lys Ile 65 70 75 80 Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile Asp Val Leu Gln Asn 85 90 95 Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln Asn Glu Val 100 105 110 Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp Leu Lys Asp 115 120 125 Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Gln 130 135 140 Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln Gln Asn Ile 145 150 155 160 Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu Leu Glu Lys 165 170 175 Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp 180 185 190 Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys Val Glu Ala Leu Gln 195 200 205 Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu Glu Asp Asn 210 215 220 Leu Lys Asp Ala Glu Thr Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly 225 230 235 240 Leu Glu Glu Ala Ile Ala Thr Lys Lys Ala Glu Leu Glu Lys Thr Gln 245 250 255 Lys Glu Leu Asp Ala Ala Leu Asn Glu Leu Gly Pro Asp Gly Asp Glu 260 265 270 Glu Glu Thr 275 2 35 PRT Escherichia coli 2 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu 35 3 21 PRT Escherichia coli 3 Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser 1 5 10 15 Leu Ile Lys His Trp 20 4 25 DNA Artificial Sequence Description of Artificial Sequence Primer 4 gcattcatga gtattcaaca tttcc 25 5 25 DNA Artificial Sequence Description of Artificial Sequence Primer 5 ccggaattct tcagcatctt ttact 25 6 45 DNA Artificial Sequence Description of Artificial Sequence Primer 6 ccggaattca tcaccatcac catcactctc ccgtagccag tcagt 45 7 30 DNA Artificial Sequence Description of Artificial Sequence Primer 7 gggaagcttc tattattcta cattattgtt 30 8 30 DNA Artificial Sequence Description of Artificial Sequence Primer 8 ccggaattct ctcccgtagc cagtcagtct 30 9 18 DNA Artificial Sequence Description of Artificial Sequence Primer 9 aaagtcgcaa tggaaggc 18 10 20 DNA Artificial Sequence Description of Artificial Sequence Primer 10 cgtagacgac gatggtcttg 20 11 23 DNA Artificial Sequence Description of Artificial Sequence Primer 11 cggaaatgat tcccttccta acg 23 12 23 DNA Artificial Sequence Description of Artificial Sequence Primer 12 tatctgcgtc gtcctacctt cag 23 13 1966 DNA Streptococcus pneumoniae CDS (1)..(1959) 13 atg aat aag aaa aaa atg att tta aca agc cta gcc agc gtc gct atc 48 Met Asn Lys Lys Lys Met Ile Leu Thr Ser Leu Ala Ser Val Ala Ile 1 5 10 15 tta ggg gct ggt ttt gtt gcg tct tcg cct act ttt gta aga gca gaa 96 Leu Gly Ala Gly Phe Val Ala Ser Ser Pro Thr Phe Val Arg Ala Glu 20 25 30 gaa gct cct gta gct aac cag tct aaa gct gag aaa gac tat gat gca 144 Glu Ala Pro Val Ala Asn Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala 35 40 45 gca gtg aaa aaa tct gaa gct gct aag aaa gat tac gaa acg gct aaa 192 Ala Val Lys Lys Ser Glu Ala Ala Lys Lys Asp Tyr Glu Thr Ala Lys 50 55 60 aag aaa gca gaa gac gct cag aag aaa tat gat gag gat cag aag aaa 240 Lys Lys Ala Glu Asp Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys 65 70 75 80 act gag gca aaa gcg gaa aaa gaa aga aaa gct tct gaa aag ata gct 288 Thr Glu Ala Lys Ala Glu Lys Glu Arg Lys Ala Ser Glu Lys Ile Ala 85 90 95 gag gca aca aaa gaa gtt caa caa gcg tac cta gct tat cta caa gct 336 Glu Ala Thr Lys Glu Val Gln Gln Ala Tyr Leu Ala Tyr Leu Gln Ala 100 105 110 agc aac gaa agt cag aga aaa gag gca gat aag aag ata aaa gaa gct 384 Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp Lys Lys Ile Lys Glu Ala 115 120 125 acg caa cgc aaa gat gag gcg gaa gct gca ttt gct act att cga aca 432 Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala Phe Ala Thr Ile Arg Thr 130 135 140 aca att gta gtt cct gaa cca agt gag tta gct gag act aag aaa aaa 480 Thr Ile Val Val Pro Glu Pro Ser Glu Leu Ala Glu Thr Lys Lys Lys 145 150 155 160 gca gaa gag gca aca aaa gaa gca gaa gta gct aag aaa aaa tct gaa 528 Ala Glu Glu Ala Thr Lys Glu Ala Glu Val Ala Lys Lys Lys Ser Glu 165 170 175 gag gca gct aaa gag gta gaa gta gag aaa aat aaa ata ctt gaa caa 576 Glu Ala Ala Lys Glu Val Glu Val Glu Lys Asn Lys Ile Leu Glu Gln 180 185 190 gat gct gaa aac gaa aag aaa att gac gta ctt caa aac aaa gtc gct 624 Asp Ala Glu Asn Glu Lys Lys Ile Asp Val Leu Gln Asn Lys Val Ala 195 200 205 gat tta gaa aaa gga att gct cct tat caa aac gaa gtc gct gaa tta 672 Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln Asn Glu Val Ala Glu Leu 210 215 220 aat aaa gaa att gct aga ctt caa agc gat tta aaa gat gct gaa gaa 720 Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp Leu Lys Asp Ala Glu Glu 225 230 235 240 aat aat gta gaa gac tac att aaa gaa ggt tta gag caa gct atc act 768 Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Gln Ala Ile Thr 245 250 255 aat aaa aaa gct gaa tta gct aca act caa caa aac ata gat aaa act 816 Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln Gln Asn Ile Asp Lys Thr 260 265 270 caa aaa gat tta gag gat gct gaa tta gaa ctt gaa aaa gta tta gct 864 Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu Leu Glu Lys Val Leu Ala 275 280 285 aca tta gac cct gaa ggt aaa act caa gat gaa tta gat aaa gaa gct 912 Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp Lys Glu Ala 290 295 300 gct gaa gct gag ttg aat gaa aaa gtt gaa gct ctt caa aac caa gtt 960 Ala Glu Ala Glu Leu Asn Glu Lys Val Glu Ala Leu Gln Asn Gln Val 305 310 315 320 gct gaa tta gaa gaa gaa ctt tca aaa ctt gaa gat aat ctt aaa gat 1008 Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu Glu Asp Asn Leu Lys Asp 325 330 335 gct gaa aca aac aac gtt gaa gac tac att aaa gaa ggt tta gaa gaa 1056 Ala Glu Thr Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Glu 340 345 350 gct atc gcg act aaa aaa gct gaa ttg gaa aaa act caa aaa gaa tta 1104 Ala Ile Ala Thr Lys Lys Ala Glu Leu Glu Lys Thr Gln Lys Glu Leu 355 360 365 gat gca gct ctt aat gag tta ggc cct gat gga gat gaa gaa gag act 1152 Asp Ala Ala Leu Asn Glu Leu Gly Pro Asp Gly Asp Glu Glu Glu Thr 370 375 380 cca gcg ccg gct cct caa cca gaa aaa cca gct gaa gag cct gag aat 1200 Pro Ala Pro Ala Pro Gln Pro Glu Lys Pro Ala Glu Glu Pro Glu Asn 385 390 395 400 cca gct cca gca cca aaa cca gag aag tca gca gat caa caa gct gaa 1248 Pro Ala Pro Ala Pro Lys Pro Glu Lys Ser Ala Asp Gln Gln Ala Glu 405 410 415 gaa gac tat gct cgt aga tca gaa gaa gaa tat aat cgc ttg acc caa 1296 Glu Asp Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg Leu Thr Gln 420 425 430 cag caa ccg cca aaa gca gaa aaa cca gct cct gca cca caa cca gag 1344 Gln Gln Pro Pro Lys Ala Glu Lys Pro Ala Pro Ala Pro Gln Pro Glu 435 440 445 caa cca gct cct gca cca aaa ata ggt tgg aaa caa gaa aac ggt atg 1392 Gln Pro Ala Pro Ala Pro Lys Ile Gly Trp Lys Gln Glu Asn Gly Met 450 455 460 tgg tac ttc tac aat act gat ggt tca atg gcg aca ggt tgg cta caa 1440 Trp Tyr Phe Tyr Asn Thr Asp Gly Ser Met Ala Thr Gly Trp Leu Gln 465 470 475 480 aac aac ggt tca tgg tac tac ctc aac agc aat ggc gct atg gct aca 1488 Asn Asn Gly Ser Trp Tyr Tyr Leu Asn Ser Asn Gly Ala Met Ala Thr 485 490 495 ggt tgg ctc caa tac aat ggt tca tgg tat tac cta aac gct aac ggc 1536 Gly Trp Leu Gln Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly 500 505 510 gct atg gcg aca ggc tgg ctc caa tac aat ggc tca tgg tac tac ctc 1584 Ala Met Ala Thr Gly Trp Leu Gln Tyr Asn Gly Ser Trp Tyr Tyr Leu 515 520 525 aac gct aac ggc gct atg gcg aca ggc tgg ctc caa tac aat ggc tca 1632 Asn Ala Asn Gly Ala Met Ala Thr Gly Trp Leu Gln Tyr Asn Gly Ser 530 535 540 tgg tac tac ctc aac gct aat ggt gat atg gcg aca gga tgg ctc caa 1680 Trp Tyr Tyr Leu Asn Ala Asn Gly Asp Met Ala Thr Gly Trp Leu Gln 545 550 555 560 tac aac ggt tca tgg tat tac ctc aac gct aat ggt gat atg gct aca 1728 Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Asp Met Ala Thr 565 570 575 ggt tgg gct aaa gtc cac ggt tca tgg tac tac ctc aac gct aac ggt 1776 Gly Trp Ala Lys Val His Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly 580 585 590 tca atg gca aca ggt tgg gtg aaa gat gga gaa acc tgg tac tat ctt 1824 Ser Met Ala Thr Gly Trp Val Lys Asp Gly Glu Thr Trp Tyr Tyr Leu 595 600 605 gaa gca tca ggt tct atg aaa gca aac caa tgg ttc caa gta tca gat 1872 Glu Ala Ser Gly Ser Met Lys Ala Asn Gln Trp Phe Gln Val Ser Asp 610 615 620 aaa tgg tac tat gtc aat ggt tta ggt tcc ctt tca gtc aac aca act 1920 Lys Trp Tyr Tyr Val Asn Gly Leu Gly Ser Leu Ser Val Asn Thr Thr 625 630 635 640 gta gat ggc tat aaa gtc aat gcc aat ggt gaa tgg gtt taagccg 1966 Val Asp Gly Tyr Lys Val Asn Ala Asn Gly Glu Trp Val 645 650 14 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 14 ccggaattcc tacaagctag caacgaaagt 30 15 35 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 15 gggaagcttt tatcaagtct cttcttcatc tccat 35 16 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 16 ccgctgcagt gtttcagcat ctttaagatt 30 17 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 17 ccgctgcaga acaacgttga agactacatt 30 18 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 18 ccgctgcagt ctcccgtagc cagtcagtct 30 19 36 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 19 ggaagcttc tattattcta cattattgtt ttcttc 36 20 2000 DNA Artificial Sequence Description of Artificial Sequence Synthetic fusion nucleotide sequence 20 ggatcttccg gaagaccttc cattctgaaa tgagctgttg acaattaatc atccggctcg 60 tataatgtgt ggaattgtga gcggataaca atttcacaca ggaaacagac c atg agt 117 Met Ser 1 att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca ttt tgc 165 Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys 5 10 15 ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa gat gct 213 Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala 20 25 30 gaa gaa ttc cta caa gct agc aac gaa agt cag aga aaa gag gca gat 261 Glu Glu Phe Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp 35 40 45 50 aag aag ata aaa gaa gct acg caa cgc aaa gat gag gcg gaa gct gca 309 Lys Lys Ile Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala 55 60 65 ttt gct act att cga aca aca att gta gtt cct gaa cca agt gag tta 357 Phe Ala Thr Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser Glu Leu 70 75 80 gct gag act aag aaa aaa gca gaa gag gca aca aaa gaa gca gaa gta 405 Ala Glu Thr Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala Glu Val 85 90 95 gct aag aaa aaa tct gaa gag gca gct aaa gag gta gaa gta gag aaa 453 Ala Lys Lys Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val Glu Lys 100 105 110 aat aaa ata ctt gaa caa gat gct gaa aac gaa aag aaa att gac gta 501 Asn Lys Ile Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile Asp Val 115 120 125 130 ctt caa aac aaa gtc gct gat tta gaa aaa gga att gct cct tat caa 549 Leu Gln Asn Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln 135 140 145 aac gaa gtc gct gaa tta aat aaa gaa att gct aga ctt caa agc gat 597 Asn Glu Val Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp 150 155 160 tta aaa gat gct gaa gaa aat aat gta gaa gac tac att aaa gaa ggt 645 Leu Lys Asp Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly 165 170 175 tta gag caa gct atc act aat aaa aaa gct gaa tta gct aca act caa 693 Leu Glu Gln Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln 180 185 190 caa aac ata gat aaa act caa aaa gat tta gag gat gct gaa tta gaa 741 Gln Asn Ile Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu 195 200 205 210 ctt gaa aaa gta tta gct aca tta gac cct gaa ggt aaa act caa gat 789 Leu Glu Lys Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp 215 220 225 gaa tta gat aaa gaa gct gct gaa gct gag ttg aat gaa aaa gtt gaa 837 Glu Leu Asp Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys Val Glu 230 235 240 gct ctt caa aac caa gtt gct gaa tta gaa gaa gaa ctt tca aaa ctt 885 Ala Leu Gln Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu 245 250 255 gaa gat aat ctt aaa gat gct gaa aca ctg cag tct ccc gta gcc agt 933 Glu Asp Asn Leu Lys Asp Ala Glu Thr Leu Gln Ser Pro Val Ala Ser 260 265 270 cag tct aaa gct gag aaa gac tat gat gca gcg aag aaa gat gct aag 981 Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala Lys Lys Asp Ala Lys 275 280 285 290 aat gcg aaa aaa gca gta gaa gat gct caa aag gct tta gat gat gca 1029 Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys Ala Leu Asp Asp Ala 295 300 305 aaa gct gct cag aaa aaa tat gac gag gat cag aag aaa act gag gag 1077 Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys Thr Glu Glu 310 315 320 aaa gcc gcg cta gaa aaa gca gcg tct gaa gag atg gat aag gca gtg 1125 Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys Ala Val 325 330 335 gca gca gtt caa caa gcg tat cta gcc tat caa caa gct aca gac aaa 1173 Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln Gln Ala Thr Asp Lys 340 345 350 gcc gca aaa gac gca gca gat aag atg ata gat gaa gct aag aaa cgc 1221 Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp Glu Ala Lys Lys Arg 355 360 365 370 gaa gaa gag gca aaa act aaa ttt aat act gtt cga gca atg gta gtt 1269 Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val Arg Ala Met Val Val 375 380 385 cct gag cca gag cag ttg gct gag act aag aaa aaa tca gaa gaa gct 1317 Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys Lys Ser Glu Glu Ala 390 395 400 aaa caa aaa gca cca gaa ctt act aaa aaa cta gaa gaa gct aaa gca 1365 Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu Glu Glu Ala Lys Ala 405 410 415 aaa tta gaa gag gct gcc gcg cta gaa aaa gca gcg tct gaa gag atg 1413 Lys Leu Glu Glu Ala Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu Met 420 425 430 gat aag gca gtg gca gca gtt caa caa gcg tat cta gcc tat caa caa 1461 Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln Gln 435 440 445 450 gct aca gac aaa gcc gca aaa gac gca gca gat aag atg ata gat gaa 1509 Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp Glu 455 460 465 gct aag aaa cgc gaa gaa gag gca aaa act aaa ttt aat act gtt cga 1557 Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val Arg 470 475 480 gca atg gta gtt cct gag cca gag cag ttg gct gag act aag aaa aaa 1605 Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys Lys 485 490 495 tca gaa gaa gct aaa caa aaa gca cca gaa ctt act aaa aaa cta gaa 1653 Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu Glu 500 505 510 gaa gct aaa gca aaa tta gaa gag gct gag aaa aaa gct act gaa gcc 1701 Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu Ala 515 520 525 530 aaa caa aaa gtg gat gct gaa gaa gtc gct cct caa gct aaa atc gct 1749 Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile Ala 535 540 545 gaa ttg gaa aat caa gtt cat aga cta gaa caa gag ctc aaa gag att 1797 Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu Ile 550 555 560 gat gag tct gaa tca gaa gat tat gct aaa gaa ggt ttc cgt gct cct 1845 Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala Pro 565 570 575 ctt caa tct aaa ttg gat gcc aaa aaa gct aaa cta tca aaa ctt gaa 1893 Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu Glu 580 585 590 gag tta agt gat aag att gat gag tta gac gct gaa att gca aaa ctt 1941 Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu Ile Ala Lys Leu 595 600 605 610 gaa gat caa ctt aaa gct gct gaa gaa aac aat aat gta gaa 1983 Glu Asp Gln Leu Lys Ala Ala Glu Glu Asn Asn Asn Val Glu 615 620 taatagaagc ttggctg 2000 21 29 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 21 gcgtcgacag cacttttaaa gttctgcta 29 22 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 22 cccaagcttt taccaatgct taatcagtga 30 23 25 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 23 gatatcacta tgcgcgaatt aaccc 25 24 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 24 aactgcaggc aactatggat gaacgaaata 30 25 290 DNA Artificial Sequence Description of Artificial Sequence Nucleotide sequence of blaSS and blaCT region of pYA3620 25 gctgttgaca attaatcatc cggctcgtat aatgtgtgga attgtgagcg gataacaatt 60 tcacacagga aacagacc atg agt att caa cat ttc cgt gtc gcc ctt att 111 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile 1 5 10 ccc ttt ttt gcg gca ttt tgc ctt cct gtt ttt gct cac cca gaa acg 159 Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr 15 20 25 ctg gtg aaa gta aaa gat gct gaa gaattcgcaa ttcccgggga tccgtcgacc 213 Leu Val Lys Val Lys Asp Ala Glu 30 35 tgcag gca act atg gat gaa cga aat aga cag atc gct gag ata ggt gcc 263 Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala 40 45 50 tca ctg att aag cat tgg taaaagctt 290 Ser Leu Ile Lys His Trp 55 26 653 PRT Streptococcus pneumoniae 26 Met Asn Lys Lys Lys Met Ile Leu Thr Ser Leu Ala Ser Val Ala Ile 1 5 10 15 Leu Gly Ala Gly Phe Val Ala Ser Ser Pro Thr Phe Val Arg Ala Glu 20 25 30 Glu Ala Pro Val Ala Asn Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala 35 40 45 Ala Val Lys Lys Ser Glu Ala Ala Lys Lys Asp Tyr Glu Thr Ala Lys 50 55 60 Lys Lys Ala Glu Asp Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys 65 70 75 80 Thr Glu Ala Lys Ala Glu Lys Glu Arg Lys Ala Ser Glu Lys Ile Ala 85 90 95 Glu Ala Thr Lys Glu Val Gln Gln Ala Tyr Leu Ala Tyr Leu Gln Ala 100 105 110 Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp Lys Lys Ile Lys Glu Ala 115 120 125 Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala Phe Ala Thr Ile Arg Thr 130 135 140 Thr Ile Val Val Pro Glu Pro Ser Glu Leu Ala Glu Thr Lys Lys Lys 145 150 155 160 Ala Glu Glu Ala Thr Lys Glu Ala Glu Val Ala Lys Lys Lys Ser Glu 165 170 175 Glu Ala Ala Lys Glu Val Glu Val Glu Lys Asn Lys Ile Leu Glu Gln 180 185 190 Asp Ala Glu Asn Glu Lys Lys Ile Asp Val Leu Gln Asn Lys Val Ala 195 200 205 Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln Asn Glu Val Ala Glu Leu 210 215 220 Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp Leu Lys Asp Ala Glu Glu 225 230 235 240 Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Gln Ala Ile Thr 245 250 255 Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln Gln Asn Ile Asp Lys Thr 260 265 270 Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu Leu Glu Lys Val Leu Ala 275 280 285 Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp Lys Glu Ala 290 295 300 Ala Glu Ala Glu Leu Asn Glu Lys Val Glu Ala Leu Gln Asn Gln Val 305 310 315 320 Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu Glu Asp Asn Leu Lys Asp 325 330 335 Ala Glu Thr Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Glu 340 345 350 Ala Ile Ala Thr Lys Lys Ala Glu Leu Glu Lys Thr Gln Lys Glu Leu 355 360 365 Asp Ala Ala Leu Asn Glu Leu Gly Pro Asp Gly Asp Glu Glu Glu Thr 370 375 380 Pro Ala Pro Ala Pro Gln Pro Glu Lys Pro Ala Glu Glu Pro Glu Asn 385 390 395 400 Pro Ala Pro Ala Pro Lys Pro Glu Lys Ser Ala Asp Gln Gln Ala Glu 405 410 415 Glu Asp Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg Leu Thr Gln 420 425 430 Gln Gln Pro Pro Lys Ala Glu Lys Pro Ala Pro Ala Pro Gln Pro Glu 435 440 445 Gln Pro Ala Pro Ala Pro Lys Ile Gly Trp Lys Gln Glu Asn Gly Met 450 455 460 Trp Tyr Phe Tyr Asn Thr Asp Gly Ser Met Ala Thr Gly Trp Leu Gln 465 470 475 480 Asn Asn Gly Ser Trp Tyr Tyr Leu Asn Ser Asn Gly Ala Met Ala Thr 485 490 495 Gly Trp Leu Gln Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly 500 505 510 Ala Met Ala Thr Gly Trp Leu Gln Tyr Asn Gly Ser Trp Tyr Tyr Leu 515 520 525 Asn Ala Asn Gly Ala Met Ala Thr Gly Trp Leu Gln Tyr Asn Gly Ser 530 535 540 Trp Tyr Tyr Leu Asn Ala Asn Gly Asp Met Ala Thr Gly Trp Leu Gln 545 550 555 560 Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Asp Met Ala Thr 565 570 575 Gly Trp Ala Lys Val His Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly 580 585 590 Ser Met Ala Thr Gly Trp Val Lys Asp Gly Glu Thr Trp Tyr Tyr Leu 595 600 605 Glu Ala Ser Gly Ser Met Lys Ala Asn Gln Trp Phe Gln Val Ser Asp 610 615 620 Lys Trp Tyr Tyr Val Asn Gly Leu Gly Ser Leu Ser Val Asn Thr Thr 625 630 635 640 Val Asp Gly Tyr Lys Val Asn Ala Asn Gly Glu Trp Val 645 650 27 32 DNA Artificial Sequence Description of Artificial Sequence Primer 27 aactgcagtt ctacattatt gttttcttca gc 32 28 524 PRT Artificial Sequence Description of Artificial Sequence Synthetic fusion protein 28 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu Glu Phe Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu 35 40 45 Ala Asp Lys Lys Ile Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu 50 55 60 Ala Ala Phe Ala Thr Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser 65 70 75 80 Glu Leu Ala Glu Thr Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala 85 90 95 Glu Val Ala Lys Lys Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val 100 105 110 Glu Lys Asn Lys Ile Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile 115 120 125 Asp Val Leu Gln Asn Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro 130 135 140 Tyr Gln Asn Glu Val Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln 145 150 155 160 Ser Asp Leu Lys Asp Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys 165 170 175 Glu Gly Leu Glu Gln Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr 180 185 190 Thr Gln Gln Asn Ile Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu 195 200 205 Leu Glu Leu Glu Lys Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr 210 215 220 Gln Asp Glu Leu Asp Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys 225 230 235 240 Val Glu Ala Leu Gln Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser 245 250 255 Lys Leu Glu Asp Asn Leu Lys Asp Ala Glu Thr Leu Gln Ser Pro Val 260 265 270 Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala Lys Lys Asp 275 280 285 Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys Ala Leu Asp 290 295 300 Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys Thr 305 310 315 320 Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys 325 330 335 Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln Gln Ala Thr 340 345 350 Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp Glu Ala Lys 355 360 365 Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val Arg Ala Met 370 375 380 Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys Lys Ser Glu 385 390 395 400 Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu Glu Glu Ala 405 410 415 Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu Ala Lys Gln 420 425 430 Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile Ala Glu Leu 435 440 445 Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu Ile Asp Glu 450 455 460 Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala Pro Leu Gln 465 470 475 480 Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu Glu Glu Leu 485 490 495 Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu Ile Ala Lys Leu Glu Asp 500 505 510 Gln Leu Lys Ala Ala Glu Glu Asn Asn Asn Val Glu 515 520 29 775 DNA Artificial Sequence Description of Artificial Sequence Nucleotide sequence of codon optimized pspARx1 29 tct ccg gta gcc agt cag tct aaa gct gag aaa gac tat gat gca gcg 48 Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala 1 5 10 15 aag aaa gat gct aag aat gct aaa aaa gca gta gaa gat gct caa aag 96 Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys 20 25 30 gct tta gat gat gca aaa gct gct cag aaa aaa tat gac gag gat cag 144 Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln 35 40 45 aag aaa act gag gag aaa gcc gcg ctg gaa aaa gca gcg tct gaa gag 192 Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu 50 55 60 atg gat aag gca gtg gca gca gtt caa caa gcg tat ctg gcc tat caa 240 Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln 65 70 75 80 caa gct aca gac aaa gcc gca aaa gac gca gca gat aag atg atc gat 288 Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp 85 90 95 gaa gct aag aaa cgc gaa gaa gag gca aaa act aaa ttt aat act gtt 336 Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val 100 105 110 cgt gca atg gta gtt cct gag cca gag cag ttg gcg gag act aag aaa 384 Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys 115 120 125 aaa tca gaa gaa gct aaa caa aaa gca cca gaa ctt act aaa aaa ctg 432 Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu 130 135 140 gaa gaa gct aaa gca aaa tta gaa gag gct gag aaa aaa gct act gaa 480 Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu 145 150 155 160 gcc aaa caa aaa gtg gat gct gaa gaa gtc gct cct caa gct aaa atc 528 Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile 165 170 175 gct gaa ttg gaa aat caa gtt cat cgt ctg gaa caa gag ctc aaa gag 576 Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu 180 185 190 att gat gag tct gaa tca gaa gat tat gct aaa gaa ggt ttc cgt gct 624 Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala 195 200 205 cct ctt caa tct aaa ttg gat gcc aaa aaa gct aaa ctg tca aaa ctt 672 Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu 210 215 220 gaa gag tta agt gat aag att gat gag tta gac gct gaa att gca aaa 720 Glu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu Ile Ala Lys 225 230 235 240 ctt gaa gat caa ctt aaa gct gct gaa gaa aac aat aat gta gaa 765 Leu Glu Asp Gln Leu Lys Ala Ala Glu Glu Asn Asn Asn Val Glu 245 250 255 taatagaagc 775 30 255 PRT Artificial Sequence Description of Artificial Sequence Protein sequence of codon optimized pspARx1 30 Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala 1 5 10 15 Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys 20 25 30 Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln 35 40 45 Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu 50 55 60 Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln 65 70 75 80 Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp 85 90 95 Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val 100 105 110 Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys 115 120 125 Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu 130 135 140 Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu 145 150 155 160 Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile 165 170 175 Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu 180 185 190 Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala 195 200 205 Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu 210 215 220 Glu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu Ile Ala Lys 225 230 235 240 Leu Glu Asp Gln Leu Lys Ala Ala Glu Glu Asn Asn Asn Val Glu 245 250 255 31 702 DNA Artificial Sequence Description of Artificial Sequence Nucleotide sequence of codon optimized pspA-EF5668-Rx1 fusion 31 ctg caa gct agc aac gaa agt cag cgt aaa gag gca gat aag aag atc 48 Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp Lys Lys Ile 1 5 10 15 aaa gaa gct acg caa cgc aaa gat gag gcg gaa gct gca ttt gct act 96 Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala Phe Ala Thr 20 25 30 att cgt aca aca att gta gtt cct gaa cca agt gag tta gct gag act 144 Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser Glu Leu Ala Glu Thr 35 40 45 aag aaa aaa gca gaa gag gca aca aaa gaa gca gaa gta gct aag aaa 192 Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala Glu Val Ala Lys Lys 50 55 60 aaa tct gaa gag gca gct aaa gag gta gaa gta gag aaa aat aaa atc 240 Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val Glu Lys Asn Lys Ile 65 70 75 80 ctt gaa caa gat gct gaa aac gaa aag aaa att gac gta ctt caa aac 288 Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile Asp Val Leu Gln Asn 85 90 95 aaa gtc gct gat tta gaa aaa ggt att gct cct tat caa aac gaa gtc 336 Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln Asn Glu Val 100 105 110 gct gaa tta aat aaa gaa att gct cgt ctt caa agc gat tta aaa gat 384 Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp Leu Lys Asp 115 120 125 gct gaa gaa aat aat gta gaa gac tac att aaa gaa ggt tta gag caa 432 Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Gln 130 135 140 gct atc act aat aaa aaa gct gaa tta gct aca act caa caa aac atc 480 Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln Gln Asn Ile 145 150 155 160 gat aaa act caa aaa gat tta gag gat gct gaa tta gaa ctt gaa aaa 528 Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu Leu Glu Lys 165 170 175 gta tta gct aca tta gac cct gaa ggt aaa act caa gat gaa tta gat 576 Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp 180 185 190 aaa gaa gct gct gaa gct gag ttg aat gaa aaa gtt gaa gct ctt caa 624 Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys Val Glu Ala Leu Gln 195 200 205 aac caa gtt gct gaa tta gaa gaa gaa ctt tca aaa ctt gaa gat aat 672 Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu Glu Asp Asn 210 215 220 ctt aaa gat gct gaa aca tgataaaagc tt 702 Leu Lys Asp Ala Glu Thr 225 230 32 230 PRT Artificial Sequence Description of Artificial Sequence Protein sequence of codon optimized pspA-EF5668-Rx1 fusion 32 Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp Lys Lys Ile 1 5 10 15 Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala Phe Ala Thr 20 25 30 Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser Glu Leu Ala Glu Thr 35 40 45 Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala Glu Val Ala Lys Lys 50 55 60 Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val Glu Lys Asn Lys Ile 65 70 75 80 Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile Asp Val Leu Gln Asn 85 90 95 Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln Asn Glu Val 100 105 110 Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp Leu Lys Asp 115 120 125 Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Gln 130 135 140 Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln Gln Asn Ile 145 150 155 160 Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu Leu Glu Lys 165 170 175 Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp 180 185 190 Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys Val Glu Ala Leu Gln 195 200 205 Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu Glu Asp Asn 210 215 220 Leu Lys Asp Ala Glu Thr 225 230 33 1473 DNA Artificial Sequence Description of Artificial Sequence Nucleotide sequence of codon optimized pspA-EF5668-Rx1 33 ctg caa gct agc aac gaa agt cag cgt aaa gag gca gat aag aag atc 48 Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp Lys Lys Ile 1 5 10 15 aaa gaa gct acg caa cgc aaa gat gag gcg gaa gct gca ttt gct act 96 Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala Phe Ala Thr 20 25 30 att cgt aca aca att gta gtt cct gaa cca agt gag tta gct gag act 144 Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser Glu Leu Ala Glu Thr 35 40 45 aag aaa aaa gca gaa gag gca aca aaa gaa gca gaa gta gct aag aaa 192 Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala Glu Val Ala Lys Lys 50 55 60 aaa tct gaa gag gca gct aaa gag gta gaa gta gag aaa aat aaa atc 240 Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val Glu Lys Asn Lys Ile 65 70 75 80 ctt gaa caa gat gct gaa aac gaa aag aaa att gac gta ctt caa aac 288 Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile Asp Val Leu Gln Asn 85 90 95 aaa gtc gct gat tta gaa aaa ggt att gct cct tat caa aac gaa gtc 336 Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln Asn Glu Val 100 105 110 gct gaa tta aat aaa gaa att gct cgt ctt caa agc gat tta aaa gat 384 Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp Leu Lys Asp 115 120 125 gct gaa gaa aat aat gta gaa gac tac att aaa gaa ggt tta gag caa 432 Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Gln 130 135 140 gct atc act aat aaa aaa gct gaa tta gct aca act caa caa aac atc 480 Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln Gln Asn Ile 145 150 155 160 gat aaa act caa aaa gat tta gag gat gct gaa tta gaa ctt gaa aaa 528 Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu Leu Glu Lys 165 170 175 gta tta gct aca tta gac cct gaa ggt aaa act caa gat gaa tta gat 576 Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp 180 185 190 aaa gaa gct gct gaa gct gag ttg aat gaa aaa gtt gaa gct ctt caa 624 Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys Val Glu Ala Leu Gln 195 200 205 aac caa gtt gct gaa tta gaa gaa gaa ctt tca aaa ctt gaa gat aat 672 Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu Glu Asp Asn 210 215 220 ctt aaa gat gct gaa aca ctg cag tct ccg gta gcc agt cag tct aaa 720 Leu Lys Asp Ala Glu Thr Leu Gln Ser Pro Val Ala Ser Gln Ser Lys 225 230 235 240 gct gag aaa gac tat gat gca gcg aag aaa gat gct aag aat gct aaa 768 Ala Glu Lys Asp Tyr Asp Ala Ala Lys Lys Asp Ala Lys Asn Ala Lys 245 250 255 aaa gca gta gaa gat gct caa aag gct tta gat gat gca aaa gct gct 816 Lys Ala Val Glu Asp Ala Gln Lys Ala Leu Asp Asp Ala Lys Ala Ala 260 265 270 cag aaa aaa tat gac gag gat cag aag aaa act gag gag aaa gcc gcg 864 Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys Thr Glu Glu Lys Ala Ala 275 280 285 ctg gaa aaa gca gcg tct gaa gag atg gat aag gca gtg gca gca gtt 912 Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys Ala Val Ala Ala Val 290 295 300 caa caa gcg tat ctg gcc tat caa caa gct aca gac aaa gcc gca aaa 960 Gln Gln Ala Tyr Leu Ala Tyr Gln Gln Ala Thr Asp Lys Ala Ala Lys 305 310 315 320 gac gca gca gat aag atg atc gat gaa gct aag aaa cgc gaa gaa gag 1008 Asp Ala Ala Asp Lys Met Ile Asp Glu Ala Lys Lys Arg Glu Glu Glu 325 330 335 gca aaa act aaa ttt aat act gtt cgt gca atg gta gtt cct gag cca 1056 Ala Lys Thr Lys Phe Asn Thr Val Arg Ala Met Val Val Pro Glu Pro 340 345 350 gag cag ttg gcg gag act aag aaa aaa tca gaa gaa gct aaa caa aaa 1104 Glu Gln Leu Ala Glu Thr Lys Lys Lys Ser Glu Glu Ala Lys Gln Lys 355 360 365 gca cca gaa ctt act aaa aaa ctg gaa gaa gct aaa gca aaa tta gaa 1152 Ala Pro Glu Leu Thr Lys Lys Leu Glu Glu Ala Lys Ala Lys Leu Glu 370 375 380 gag gct gag aaa aaa gct act gaa gcc aaa caa aaa gtg gat gct gaa 1200 Glu Ala Glu Lys Lys Ala Thr Glu Ala Lys Gln Lys Val Asp Ala Glu 385 390 395 400 gaa gtc gct cct caa gct aaa atc gct gaa ttg gaa aat caa gtt cat 1248 Glu Val Ala Pro Gln Ala Lys Ile Ala Glu Leu Glu Asn Gln Val His 405 410 415 cgt ctg gaa caa gag ctc aaa gag att gat gag tct gaa tca gaa gat 1296 Arg Leu Glu Gln Glu Leu Lys Glu Ile Asp Glu Ser Glu Ser Glu Asp 420 425 430 tat gct aaa gaa ggt ttc cgt gct cct ctt caa tct aaa ttg gat gcc 1344 Tyr Ala Lys Glu Gly Phe Arg Ala Pro Leu Gln Ser Lys Leu Asp Ala 435 440 445 aaa aaa gct aaa ctg tca aaa ctt gaa gag tta agt gat aag att gat 1392 Lys Lys Ala Lys Leu Ser Lys Leu Glu Glu Leu Ser Asp Lys Ile Asp 450 455 460 gag tta gac gct gaa att gca aaa ctt gaa gat caa ctt aaa gct gct 1440 Glu Leu Asp Ala Glu Ile Ala Lys Leu Glu Asp Gln Leu Lys Ala Ala 465 470 475 480 gaa gaa aac aat aat gta gaa taatagaagc tt 1473 Glu Glu Asn Asn Asn Val Glu 485 34 487 PRT Artificial Sequence Description of Artificial Sequence Protein sequence of codon optimized pspA-EF5668-Rx1 34 Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu Ala Asp Lys Lys Ile 1 5 10 15 Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu Ala Ala Phe Ala Thr 20 25 30 Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser Glu Leu Ala Glu Thr 35 40 45 Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala Glu Val Ala Lys Lys 50 55 60 Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val Glu Lys Asn Lys Ile 65 70 75 80 Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile Asp Val Leu Gln Asn 85 90 95 Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro Tyr Gln Asn Glu Val 100 105 110 Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln Ser Asp Leu Lys Asp 115 120 125 Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Gln 130 135 140 Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr Thr Gln Gln Asn Ile 145 150 155 160 Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu Leu Glu Leu Glu Lys 165 170 175 Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp 180 185 190 Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys Val Glu Ala Leu Gln 195 200 205 Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser Lys Leu Glu Asp Asn 210 215 220 Leu Lys Asp Ala Glu Thr Leu Gln Ser Pro Val Ala Ser Gln Ser Lys 225 230 235 240 Ala Glu Lys Asp Tyr Asp Ala Ala Lys Lys Asp Ala Lys Asn Ala Lys 245 250 255 Lys Ala Val Glu Asp Ala Gln Lys Ala Leu Asp Asp Ala Lys Ala Ala 260 265 270 Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys Thr Glu Glu Lys Ala Ala 275 280 285 Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys Ala Val Ala Ala Val 290 295 300 Gln Gln Ala Tyr Leu Ala Tyr Gln Gln Ala Thr Asp Lys Ala Ala Lys 305 310 315 320 Asp Ala Ala Asp Lys Met Ile Asp Glu Ala Lys Lys Arg Glu Glu Glu 325 330 335 Ala Lys Thr Lys Phe Asn Thr Val Arg Ala Met Val Val Pro Glu Pro 340 345 350 Glu Gln Leu Ala Glu Thr Lys Lys Lys Ser Glu Glu Ala Lys Gln Lys 355 360 365 Ala Pro Glu Leu Thr Lys Lys Leu Glu Glu Ala Lys Ala Lys Leu Glu 370 375 380 Glu Ala Glu Lys Lys Ala Thr Glu Ala Lys Gln Lys Val Asp Ala Glu 385 390 395 400 Glu Val Ala Pro Gln Ala Lys Ile Ala Glu Leu Glu Asn Gln Val His 405 410 415 Arg Leu Glu Gln Glu Leu Lys Glu Ile Asp Glu Ser Glu Ser Glu Asp 420 425 430 Tyr Ala Lys Glu Gly Phe Arg Ala Pro Leu Gln Ser Lys Leu Asp Ala 435 440 445 Lys Lys Ala Lys Leu Ser Lys Leu Glu Glu Leu Ser Asp Lys Ile Asp 450 455 460 Glu Leu Asp Ala Glu Ile Ala Lys Leu Glu Asp Gln Leu Lys Ala Ala 465 470 475 480 Glu Glu Asn Asn Asn Val Glu 485 35 45 DNA Artificial Sequence Description of Artificial Sequence Primer 35 ggcgaattcc tgcagtctcc ggtagccagt cagtctaaag ctgag 45 36 159 DNA Artificial Sequence Description of Artificial Sequence Primer 36 ggcaagcttt tattctacat tattgttttc ttcagcagct ttaagttgat cttcaagttt 60 tgcaatttca gcgtctaact catcaatctt atcacttaac tcttcaagtt ttgacagttt 120 agcttttttg gcatccaatt tagattgaag aggagcacg 159 37 33 DNA Artificial Sequence Description of Artificial Sequence Primer 37 gccgcgctgg aaaaagcagc gtctgaagag atg 33 38 33 DNA Artificial Sequence Description of Artificial Sequence Primer 38 gccgcgcggc tttctcctca gttttcttct gat 33 39 30 DNA Artificial Sequence Description of Artificial Sequence Primer 39 ggcatcgatg aagctaagaa acgcgaagaa 30 40 87 DNA Artificial Sequence Description of Artificial Sequence Primer 40 gccatcgatc atcttatctg ctgcgtcttt tgcggctttg tctgtagctt gttgataggc 60 cagatacgct tgttgaactg ctgccac 87 41 45 DNA Artificial Sequence Description of Artificial Sequence Primer 41 ggcaaattta atactgttcg tgcaatggta gttcctgagc cagag 45 42 30 DNA Artificial Sequence Description of Artificial Sequence Primer 42 gccaaattta gtttttgcct cttcttcgcg 30 43 57 DNA Artificial Sequence Description of Artificial Sequence Primer 43 aaacaaaaag caccagaact tactaaaaaa ctggaagaag ctaaagcaaa attagaa 57 44 39 DNA Artificial Sequence Description of Artificial Sequence Primer 44 tttagcttct tccagttttt tagtaagttc tggtgcttt 39 45 30 DNA Artificial Sequence Description of Artificial Sequence Primer 45 ggcgagctca aagagattga tgagtctgaa 30 46 48 DNA Artificial Sequence Description of Artificial Sequence Primer 46 aaagagctct tgttccagac gatgaacttg attttccaat tcagcgat 48 47 80 DNA Artificial Sequence Description of Artificial Sequence Primer 47 gaagaattcc tgcaagctag caacgaaagt cagcgtaaag aggcagataa gaagatcaaa 60 gaagctacgc aacgcaaaga 80 48 60 DNA Artificial Sequence Description of Artificial Sequence Primer 48 ggcaagcttc tgcagagtct cttcttcatc accatcaggg cctaactcat taagagctgc 60 49 30 DNA Artificial Sequence Description of Artificial Sequence Primer 49 ggccaattgt agttcctgaa ccaagtgagt 30 50 30 DNA Artificial Sequence Description of Artificial Sequence Primer 50 ggccaattgt tgtacgaata gtagcaaatg 30 51 72 DNA Artificial Sequence Description of Artificial Sequence Primer 51 ggcgtacgtc aattttcttt tcgttttcag catcttgttc aaggatttta tttttctcta 60 cttctacctc tt 72 52 60 DNA Artificial Sequence Description of Artificial Sequence Primer 52 ggcgtacttc aaaacaaagt cgctgattta gaaaaaggta ttgctcctta tcaaaacgaa 60 53 30 DNA Artificial Sequence Description of Artificial Sequence Primer 53 ggctttaaaa gatgctgaag aaaataatgt 30 54 48 DNA Artificial Sequence Description of Artificial Sequence Primer 54 ggctttaaat cgctttgaag acgagcaatt tctttattta attcagcg 48 55 30 DNA Artificial Sequence Description of Artificial Sequence Primer 55 ggcatcgata aaactcaaaa agatttagag 30 56 30 DNA Artificial Sequence Description of Artificial Sequence Primer 56 ggcatcgatg ttttgttgag ttgtagctaa 30 57 30 DNA Artificial Sequence Description of Artificial Sequence Primer 57 ccggaattcc tacaagctag caacgaaagt 30 58 35 DNA Artificial Sequence Description of Artificial Sequence Primer 58 ccgaagcttc tattatgttt cagcatcttt aagat 35 59 37 DNA Artificial Sequence Description of Artificial Sequence Primer 59 aagcttctgc agtgtttcag catctttaag attatct 37 60 35 DNA Artificial Sequence Description of Artificial Sequence Primer 60 ccgaagcttc tattatgttt cagcatcttt aagat 35 61 25 DNA Artificial Sequence Description of Artificial Sequence Primer 61 gtataatgtg tggaattgtg agcgg 25 62 30 DNA Artificial Sequence Description of Artificial Sequence Primer 62 ccggaattct ctcccgtagc cagtcagtct 30 63 96 DNA Artificial Sequence Description of Artificial Sequence Primer 63 ggcaagctat tattctacat tattgttttc ttcagcagct ttaagttgat cttcaagttt 60 tgcaatttca gcgtctaact catcaatctt atcact 96 64 900 DNA Artificial Sequence Description of Artificial Sequence Nucleotide sequence of blaSS-pspA-EF5668-bla C-term region in pYA3637 64 atg agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca 48 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 ttt tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa 96 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 gat gct gaa gaa ttc ctg caa gct agc aac gaa agt cag cgt aaa gag 144 Asp Ala Glu Glu Phe Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu 35 40 45 gca gat aag aag atc aaa gaa gct acg caa cgc aaa gat gag gcg gaa 192 Ala Asp Lys Lys Ile Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu 50 55 60 gct gca ttt gct act att cgt aca aca att gta gtt cct gaa cca agt 240 Ala Ala Phe Ala Thr Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser 65 70 75 80 gag tta gct gag act aag aaa aaa gca gaa gag gca aca aaa gaa gca 288 Glu Leu Ala Glu Thr Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala 85 90 95 gaa gta gct aag aaa aaa tct gaa gag gca gct aaa gag gta gaa gta 336 Glu Val Ala Lys Lys Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val 100 105 110 gag aaa aat aaa atc ctt gaa caa gat gct gaa aac gaa aag aaa att 384 Glu Lys Asn Lys Ile Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile 115 120 125 gac gta ctt caa aac aaa gtc gct gat tta gaa aaa ggt att gct cct 432 Asp Val Leu Gln Asn Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro 130 135 140 tat caa aac gaa gtc gct gaa tta aat aaa gaa att gct cgt ctt caa 480 Tyr Gln Asn Glu Val Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln 145 150 155 160 agc gat tta aaa gat gct gaa gaa aat aat gta gaa gac tac att aaa 528 Ser Asp Leu Lys Asp Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys 165 170 175 gaa ggt tta gag caa gct atc act aat aaa aaa gct gaa tta gct aca 576 Glu Gly Leu Glu Gln Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr 180 185 190 act caa caa aac atc gat aaa act caa aaa gat tta gag gat gct gaa 624 Thr Gln Gln Asn Ile Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu 195 200 205 tta gaa ctt gaa aaa gta tta gct aca tta gac cct gaa ggt aaa act 672 Leu Glu Leu Glu Lys Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr 210 215 220 caa gat gaa tta gat aaa gaa gct gct gaa gct gag ttg aat gaa aaa 720 Gln Asp Glu Leu Asp Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys 225 230 235 240 gtt gaa gct ctt caa aac caa gtt gct gaa tta gaa gaa gaa ctt tca 768 Val Glu Ala Leu Gln Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser 245 250 255 aaa ctt gaa gat aat ctt aaa gat gct gaa aca ctg cag gca act atg 816 Lys Leu Glu Asp Asn Leu Lys Asp Ala Glu Thr Leu Gln Ala Thr Met 260 265 270 gat gaa cga aat aga cag atc gct gag ata ggt gcc tca ctg att aag 864 Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys 275 280 285 cat tgg taaaagcttg gctgttttgg cggatgagag 900 His Trp 290 65 290 PRT Artificial Sequence Description of Artificial Sequence Protein sequence of blaSS-pspA-EF5668-bla C-term region in pYA3637 65 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu Glu Phe Leu Gln Ala Ser Asn Glu Ser Gln Arg Lys Glu 35 40 45 Ala Asp Lys Lys Ile Lys Glu Ala Thr Gln Arg Lys Asp Glu Ala Glu 50 55 60 Ala Ala Phe Ala Thr Ile Arg Thr Thr Ile Val Val Pro Glu Pro Ser 65 70 75 80 Glu Leu Ala Glu Thr Lys Lys Lys Ala Glu Glu Ala Thr Lys Glu Ala 85 90 95 Glu Val Ala Lys Lys Lys Ser Glu Glu Ala Ala Lys Glu Val Glu Val 100 105 110 Glu Lys Asn Lys Ile Leu Glu Gln Asp Ala Glu Asn Glu Lys Lys Ile 115 120 125 Asp Val Leu Gln Asn Lys Val Ala Asp Leu Glu Lys Gly Ile Ala Pro 130 135 140 Tyr Gln Asn Glu Val Ala Glu Leu Asn Lys Glu Ile Ala Arg Leu Gln 145 150 155 160 Ser Asp Leu Lys Asp Ala Glu Glu Asn Asn Val Glu Asp Tyr Ile Lys 165 170 175 Glu Gly Leu Glu Gln Ala Ile Thr Asn Lys Lys Ala Glu Leu Ala Thr 180 185 190 Thr Gln Gln Asn Ile Asp Lys Thr Gln Lys Asp Leu Glu Asp Ala Glu 195 200 205 Leu Glu Leu Glu Lys Val Leu Ala Thr Leu Asp Pro Glu Gly Lys Thr 210 215 220 Gln Asp Glu Leu Asp Lys Glu Ala Ala Glu Ala Glu Leu Asn Glu Lys 225 230 235 240 Val Glu Ala Leu Gln Asn Gln Val Ala Glu Leu Glu Glu Glu Leu Ser 245 250 255 Lys Leu Glu Asp Asn Leu Lys Asp Ala Glu Thr Leu Gln Ala Thr Met 260 265 270 Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys 275 280 285 His Trp 290 66 932 DNA Streptococcus pneumoniae CDS (1)..(927) 66 atg aaa aaa tta ggt aca tta ctc gtt ctc ttt ctt tct gca atc att 48 Met Lys Lys Leu Gly Thr Leu Leu Val Leu Phe Leu Ser Ala Ile Ile 1 5 10 15 ctt gta gca tgt gct agc gga aaa aaa gat aca act tct ggt caa aaa 96 Leu Val Ala Cys Ala Ser Gly Lys Lys Asp Thr Thr Ser Gly Gln Lys 20 25 30 cta aaa gtt gtt gct aca aac tca atc atc gct gat att act aaa aat 144 Leu Lys Val Val Ala Thr Asn Ser Ile Ile Ala Asp Ile Thr Lys Asn 35 40 45 att gct ggt gac aaa att gac ctt cat agt atc gtt ccg att ggg caa 192 Ile Ala Gly Asp Lys Ile Asp Leu His Ser Ile Val Pro Ile Gly Gln 50 55 60 gac cca cac gaa tac gaa cca ctt cct gaa gac gtt aag aaa act tct 240 Asp Pro His Glu Tyr Glu Pro Leu Pro Glu Asp Val Lys Lys Thr Ser 65 70 75 80 gag gct gat ttg att ttc tat aac ggt atc aac ctt gaa aca ggt ggc 288 Glu Ala Asp Leu Ile Phe Tyr Asn Gly Ile Asn Leu Glu Thr Gly Gly 85 90 95 aat gct tgg ttt aca aaa ttg gta gaa aat gcc aag aaa act gaa aac 336 Asn Ala Trp Phe Thr Lys Leu Val Glu Asn Ala Lys Lys Thr Glu Asn 100 105 110 aaa gac tac ttc gca gtc agc gac ggc gtt gat gtt atc tac ctt gaa 384 Lys Asp Tyr Phe Ala Val Ser Asp Gly Val Asp Val Ile Tyr Leu Glu 115 120 125 ggt caa aat gaa aaa gga aaa gaa gac cca cac gct tgg ctt aac ctt 432 Gly Gln Asn Glu Lys Gly Lys Glu Asp Pro His Ala Trp Leu Asn Leu 130 135 140 gaa aac ggt att att ttt gct aaa aat atc gcc aaa caa ttg agc gcc 480 Glu Asn Gly Ile Ile Phe Ala Lys Asn Ile Ala Lys Gln Leu Ser Ala 145 150 155 160 aaa gac cct aac aat aaa gaa ttc tat gaa aaa aat ctc aaa gaa tat 528 Lys Asp Pro Asn Asn Lys Glu Phe Tyr Glu Lys Asn Leu Lys Glu Tyr 165 170 175 act gat aag tta gac aaa ctt gat aaa gaa agt aag gat aaa ttt aat 576 Thr Asp Lys Leu Asp Lys Leu Asp Lys Glu Ser Lys Asp Lys Phe Asn 180 185 190 aag atc cct gct gaa aag aaa ctc att gta acc agc gaa gga gca ttc 624 Lys Ile Pro Ala Glu Lys Lys Leu Ile Val Thr Ser Glu Gly Ala Phe 195 200 205 aaa tac ttc tct aaa gcc tat ggt gtc cca agt gcc tac atc tgg gaa 672 Lys Tyr Phe Ser Lys Ala Tyr Gly Val Pro Ser Ala Tyr Ile Trp Glu 210 215 220 atc aat act gaa gaa gaa gga act cct gaa caa atc aag acc ttg gtt 720 Ile Asn Thr Glu Glu Glu Gly Thr Pro Glu Gln Ile Lys Thr Leu Val 225 230 235 240 gaa aaa ctt cgc caa aca aaa gtt cca tca ctc ttt gta gaa tca agt 768 Glu Lys Leu Arg Gln Thr Lys Val Pro Ser Leu Phe Val Glu Ser Ser 245 250 255 gtg gat gac cgt cca atg aaa act gtt tct caa gac aca aac atc cca 816 Val Asp Asp Arg Pro Met Lys Thr Val Ser Gln Asp Thr Asn Ile Pro 260 265 270 atc tac gca caa atc ttt act gac tct atc gca gaa caa ggt aaa gaa 864 Ile Tyr Ala Gln Ile Phe Thr Asp Ser Ile Ala Glu Gln Gly Lys Glu 275 280 285 ggc gac agc tac tac agc atg atg aaa tac aac ctt gac aag att gct 912 Gly Asp Ser Tyr Tyr Ser Met Met Lys Tyr Asn Leu Asp Lys Ile Ala 290 295 300 gaa gga ttg gca aaa taagc 932 Glu Gly Leu Ala Lys 305 67 309 PRT Streptococcus pneumoniae 67 Met Lys Lys Leu Gly Thr Leu Leu Val Leu Phe Leu Ser Ala Ile Ile 1 5 10 15 Leu Val Ala Cys Ala Ser Gly Lys Lys Asp Thr Thr Ser Gly Gln Lys 20 25 30 Leu Lys Val Val Ala Thr Asn Ser Ile Ile Ala Asp Ile Thr Lys Asn 35 40 45 Ile Ala Gly Asp Lys Ile Asp Leu His Ser Ile Val Pro Ile Gly Gln 50 55 60 Asp Pro His Glu Tyr Glu Pro Leu Pro Glu Asp Val Lys Lys Thr Ser 65 70 75 80 Glu Ala Asp Leu Ile Phe Tyr Asn Gly Ile Asn Leu Glu Thr Gly Gly 85 90 95 Asn Ala Trp Phe Thr Lys Leu Val Glu Asn Ala Lys Lys Thr Glu Asn 100 105 110 Lys Asp Tyr Phe Ala Val Ser Asp Gly Val Asp Val Ile Tyr Leu Glu 115 120 125 Gly Gln Asn Glu Lys Gly Lys Glu Asp Pro His Ala Trp Leu Asn Leu 130 135 140 Glu Asn Gly Ile Ile Phe Ala Lys Asn Ile Ala Lys Gln Leu Ser Ala 145 150 155 160 Lys Asp Pro Asn Asn Lys Glu Phe Tyr Glu Lys Asn Leu Lys Glu Tyr 165 170 175 Thr Asp Lys Leu Asp Lys Leu Asp Lys Glu Ser Lys Asp Lys Phe Asn 180 185 190 Lys Ile Pro Ala Glu Lys Lys Leu Ile Val Thr Ser Glu Gly Ala Phe 195 200 205 Lys Tyr Phe Ser Lys Ala Tyr Gly Val Pro Ser Ala Tyr Ile Trp Glu 210 215 220 Ile Asn Thr Glu Glu Glu Gly Thr Pro Glu Gln Ile Lys Thr Leu Val 225 230 235 240 Glu Lys Leu Arg Gln Thr Lys Val Pro Ser Leu Phe Val Glu Ser Ser 245 250 255 Val Asp Asp Arg Pro Met Lys Thr Val Ser Gln Asp Thr Asn Ile Pro 260 265 270 Ile Tyr Ala Gln Ile Phe Thr Asp Ser Ile Ala Glu Gln Gly Lys Glu 275 280 285 Gly Asp Ser Tyr Tyr Ser Met Met Lys Tyr Asn Leu Asp Lys Ile Ala 290 295 300 Glu Gly Leu Ala Lys 305 68 142 DNA Artificial Sequence Description of Artificial Sequence Nucleotide sequence of Ptrc promoter region 68 aatgagctgt tgacaattaa tcatccggct cgtataatgt gtgggtggaa ttgtgagcgg 60 ataacaattt cacacaggaa acagacc atg gga att cgc aat tcc cgg gga tcc 114 Met Gly Ile Arg Asn Ser Arg Gly Ser 1 5 gtc gac ctg cag cca agc tcc caa gct t 142 Val Asp Leu Gln Pro Ser Ser Gln Ala 10 15 69 18 PRT Artificial Sequence Description of Artificial Sequence Peptide sequence of Ptrc promoter region 69 Met Gly Ile Arg Asn Ser Arg Gly Ser Val Asp Leu Gln Pro Ser Ser 1 5 10 15 Gln Ala 70 243 DNA Artificial Sequence Description of Artificial Sequence Beta-lactamase signal sequence from pBR322 70 aatgagctgt tgacaattaa tcatccggct cgtataatgt gtgggtggaa ttgtgagcgg 60 ataacaattt cacacaggaa acagacc atg agt att caa cat ttc cgt gtc gcc 114 Met Ser Ile Gln His Phe Arg Val Ala 1 5 ctt att ccc ttt ttt gcg gca ttt tgc ctt cct gtt ttt gct cac cca 162 Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro 10 15 20 25 gaa acg ctg gtg aaa gta aaa gat gct gaa gaattcgcaa ttcccgggga 212 Glu Thr Leu Val Lys Val Lys Asp Ala Glu 30 35 tccgtcgacc tgcagccaag ctcccaagct t 243 71 1860 DNA Streptococcus pneumoniae CDS (1)..(1857) 71 atg aat aag aaa aaa atg att tta aca agt cta gcc agc gtc gct atc 48 Met Asn Lys Lys Lys Met Ile Leu Thr Ser Leu Ala Ser Val Ala Ile 1 5 10 15 tta ggg gct ggt ttt gtt gcg tct cag cct act gtt gta aga gca gaa 96 Leu Gly Ala Gly Phe Val Ala Ser Gln Pro Thr Val Val Arg Ala Glu 20 25 30 gaa tct ccc gta gcc agt cag tct aaa gct gag aaa gac tat gat gca 144 Glu Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala 35 40 45 gcg aag aaa gat gct aag aat gcg aaa aaa gca gta gaa gat gct caa 192 Ala Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln 50 55 60 aag gct tta gat gat gca aaa gct gct cag aaa aaa tat gac gag gat 240 Lys Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp 65 70 75 80 cag aag aaa act gag gag aaa gcc gcg cta gaa aaa gca gcg tct gaa 288 Gln Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu 85 90 95 gag atg gat aag gca gtg gca gca gtt caa caa gcg tat cta gcc tat 336 Glu Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr 100 105 110 caa caa gct aca gac aaa gcc gca aaa gac gca gca gat aag atg ata 384 Gln Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile 115 120 125 gat gaa gct aag aaa cgc gaa gaa gag gca aaa act aaa ttt aat act 432 Asp Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr 130 135 140 gtt cga gca atg gta gtt cct gag cca gag cag ttg gct gag act aag 480 Val Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys 145 150 155 160 aaa aaa tca gaa gaa gct aaa caa aaa gca cca gaa ctt act aaa aaa 528 Lys Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys 165 170 175 cta gaa gaa gct aaa gca aaa tta gaa gag gct gag aaa aaa gct act 576 Leu Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr 180 185 190 gaa gcc aaa caa aaa gtg gat gct gaa gaa gtc gct cct caa gct aaa 624 Glu Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys 195 200 205 atc gct gaa ttg gaa aat caa gtt cat aga cta gaa caa gag ctc aaa 672 Ile Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys 210 215 220 gag att gat gag tct gaa tca gaa gat tat gct aaa gaa ggt ttc cgt 720 Glu Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg 225 230 235 240 gct cct ctt caa tct aaa ttg gat gcc aaa aaa gct aaa cta tca aaa 768 Ala Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys 245 250 255 ctt gaa gag tta agt gat aag att gat gag tta gac gct gaa att gca 816 Leu Glu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu Ile Ala 260 265 270 aaa ctt gaa gat caa ctt aaa gct gct gaa gaa aac aat aat gta gaa 864 Lys Leu Glu Asp Gln Leu Lys Ala Ala Glu Glu Asn Asn Asn Val Glu 275 280 285 gac tac ttt aaa gaa ggt tta gag aaa act att gct gct aaa aaa gct 912 Asp Tyr Phe Lys Glu Gly Leu Glu Lys Thr Ile Ala Ala Lys Lys Ala 290 295 300 gaa tta gaa aaa act gaa gct gac ctt aag aaa gca gtt aat gag cca 960 Glu Leu Glu Lys Thr Glu Ala Asp Leu Lys Lys Ala Val Asn Glu Pro 305 310 315 320 gaa aaa cca gct cca gct cca gaa act cca gcc cca gaa gca cca gct 1008 Glu Lys Pro Ala Pro Ala Pro Glu Thr Pro Ala Pro Glu Ala Pro Ala 325 330 335 gaa caa cca aaa cca gcg ccg gct cct caa cca gct ccc gca cca aaa 1056 Glu Gln Pro Lys Pro Ala Pro Ala Pro Gln Pro Ala Pro Ala Pro Lys 340 345 350 cca gag aag cca gct gaa caa cca aaa cca gaa aaa aca gat gat caa 1104 Pro Glu Lys Pro Ala Glu Gln Pro Lys Pro Glu Lys Thr Asp Asp Gln 355 360 365 caa gct gaa gaa gac tat gct cgt aga tca gaa gaa gaa tat aat cgc 1152 Gln Ala Glu Glu Asp Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg 370 375 380 ttg act caa cag caa ccg cca aaa gct gaa aaa cca gct cct gca cca 1200 Leu Thr Gln Gln Gln Pro Pro Lys Ala Glu Lys Pro Ala Pro Ala Pro 385 390 395 400 aaa aca ggc tgg aaa caa gaa aac ggt atg tgg tac ttc tac aat act 1248 Lys Thr Gly Trp Lys Gln Glu Asn Gly Met Trp Tyr Phe Tyr Asn Thr 405 410 415 gat ggt tca atg gcg aca gga tgg ctc caa aac aac ggt tca tgg tac 1296 Asp Gly Ser Met Ala Thr Gly Trp Leu Gln Asn Asn Gly Ser Trp Tyr 420 425 430 tac ctc aac agc aat ggt gct atg gct aca ggt tgg ctc caa tac aat 1344 Tyr Leu Asn Ser Asn Gly Ala Met Ala Thr Gly Trp Leu Gln Tyr Asn 435 440 445 ggt tca tgg tat tac ctc aac gct aac ggc gct atg gca aca ggt tgg 1392 Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met Ala Thr Gly Trp 450 455 460 gct aaa gtc aac ggt tca tgg tac tac ctc aac gct aat ggt gct atg 1440 Ala Lys Val Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met 465 470 475 480 gct aca ggt tgg ctc caa tac aac ggt tca tgg tat tac ctc aac gct 1488 Ala Thr Gly Trp Leu Gln Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala 485 490 495 aac ggc gct atg gca aca ggt tgg gct aaa gtc aac ggt tca tgg tac 1536 Asn Gly Ala Met Ala Thr Gly Trp Ala Lys Val Asn Gly Ser Trp Tyr 500 505 510 tac ctc aac gct aat ggt gct atg gct aca ggt tgg ctc caa tac aac 1584 Tyr Leu Asn Ala Asn Gly Ala Met Ala Thr Gly Trp Leu Gln Tyr Asn 515 520 525 ggt tca tgg tac tac ctc aac gct aac ggt gct atg gct aca ggt tgg 1632 Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met Ala Thr Gly Trp 530 535 540 gct aaa gtc aac ggt tca tgg tac tac ctc aac gct aat ggt gct atg 1680 Ala Lys Val Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met 545 550 555 560 gca aca ggt tgg gtg aaa gat gga gat acc tgg tac tat ctt gaa gca 1728 Ala Thr Gly Trp Val Lys Asp Gly Asp Thr Trp Tyr Tyr Leu Glu Ala 565 570 575 tca ggt gct atg aaa gca agc caa tgg ttc aaa gta tca gat aaa tgg 1776 Ser Gly Ala Met Lys Ala Ser Gln Trp Phe Lys Val Ser Asp Lys Trp 580 585 590 tac tat gtc aat ggt tta ggt gcc ctt gca gtc aac aca act gta gat 1824 Tyr Tyr Val Asn Gly Leu Gly Ala Leu Ala Val Asn Thr Thr Val Asp 595 600 605 ggc tat aaa gtc aat gcc aat ggt gaa tgg gtt taa 1860 Gly Tyr Lys Val Asn Ala Asn Gly Glu Trp Val 610 615 72 619 PRT Streptococcus pneumoniae 72 Met Asn Lys Lys Lys Met Ile Leu Thr Ser Leu Ala Ser Val Ala Ile 1 5 10 15 Leu Gly Ala Gly Phe Val Ala Ser Gln Pro Thr Val Val Arg Ala Glu 20 25 30 Glu Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala 35 40 45 Ala Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln 50 55 60 Lys Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp 65 70 75 80 Gln Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu 85 90 95 Glu Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr 100 105 110 Gln Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile 115 120 125 Asp Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr 130 135 140 Val Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys 145 150 155 160 Lys Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys 165 170 175 Leu Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr 180 185 190 Glu Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys 195 200 205 Ile Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys 210 215 220 Glu Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg 225 230 235 240 Ala Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys 245 250 255 Leu Glu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu Ile Ala 260 265 270 Lys Leu Glu Asp Gln Leu Lys Ala Ala Glu Glu Asn Asn Asn Val Glu 275 280 285 Asp Tyr Phe Lys Glu Gly Leu Glu Lys Thr Ile Ala Ala Lys Lys Ala 290 295 300 Glu Leu Glu Lys Thr Glu Ala Asp Leu Lys Lys Ala Val Asn Glu Pro 305 310 315 320 Glu Lys Pro Ala Pro Ala Pro Glu Thr Pro Ala Pro Glu Ala Pro Ala 325 330 335 Glu Gln Pro Lys Pro Ala Pro Ala Pro Gln Pro Ala Pro Ala Pro Lys 340 345 350 Pro Glu Lys Pro Ala Glu Gln Pro Lys Pro Glu Lys Thr Asp Asp Gln 355 360 365 Gln Ala Glu Glu Asp Tyr Ala Arg Arg Ser Glu Glu Glu Tyr Asn Arg 370 375 380 Leu Thr Gln Gln Gln Pro Pro Lys Ala Glu Lys Pro Ala Pro Ala Pro 385 390 395 400 Lys Thr Gly Trp Lys Gln Glu Asn Gly Met Trp Tyr Phe Tyr Asn Thr 405 410 415 Asp Gly Ser Met Ala Thr Gly Trp Leu Gln Asn Asn Gly Ser Trp Tyr 420 425 430 Tyr Leu Asn Ser Asn Gly Ala Met Ala Thr Gly Trp Leu Gln Tyr Asn 435 440 445 Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met Ala Thr Gly Trp 450 455 460 Ala Lys Val Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met 465 470 475 480 Ala Thr Gly Trp Leu Gln Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala 485 490 495 Asn Gly Ala Met Ala Thr Gly Trp Ala Lys Val Asn Gly Ser Trp Tyr 500 505 510 Tyr Leu Asn Ala Asn Gly Ala Met Ala Thr Gly Trp Leu Gln Tyr Asn 515 520 525 Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met Ala Thr Gly Trp 530 535 540 Ala Lys Val Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met 545 550 555 560 Ala Thr Gly Trp Val Lys Asp Gly Asp Thr Trp Tyr Tyr Leu Glu Ala 565 570 575 Ser Gly Ala Met Lys Ala Ser Gln Trp Phe Lys Val Ser Asp Lys Trp 580 585 590 Tyr Tyr Val Asn Gly Leu Gly Ala Leu Ala Val Asn Thr Thr Val Asp 595 600 605 Gly Tyr Lys Val Asn Ala Asn Gly Glu Trp Val 610 615 

What is claimed is:
 1. A vaccine comprising a live attenuated strain of pathogenic gram negative bacteria, wherein (a) the strain of bacteria comprises a first polynucleotide that encodes an antigen, (b) the first polynucleotide is operably linked to a second polynucleotide that encodes a secretion peptide, (c) the antigen is from a source that is different than the live attenuated strain of bacteria, and (d) the vaccine elicits a Th2-type immune response in a vertebrate.
 2. The vaccine of claim 1 further comprising a balanced-lethal host-vector system.
 3. The vaccine of claim 2 further comprising an environmental limitation viability system.
 4. The vaccine of claim 2 wherein the live attenuated strain of bacteria comprises an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and a plasmid, wherein the plasmid comprises a polynucleotide that encodes a functional Asd and the first and second polynucleotides.
 5. The vaccine of claim 1 wherein the strain of bacteria is a member of a group of bacteria selected from the group consisting of Enterobacteriaceae, Vibrionaceae, Francisellaceae, Legionallales, Pseudomonadacea and Pasteurellaceae.
 6. The vaccine of claim 5 wherein the strain of bacteria is a member of the Enterobacteriaceae group.
 7. The vaccine of claim 6 wherein the strain of bacteria is selected from the group consisting of Salmonella, Escherichia, Shigella and Yersinia.
 8. The vaccine of claim 7 wherein the strain of bacteria is a Salmonella enterica.
 9. The vaccine of claim 8 wherein the Salmonella enterica comprises an attenuating mutation in a gene encoding a protein selected from the group consisting of cyclic AMP receptor protein (“Crp”), adenylate cyclase (“Cya”), aspartate β-semialdehyde dehydrogenase (“Asd”) and DNA adenine methylase (“Dam”).
 10. The vaccine of claim 9 wherein the protein is cyclic AMP receptor protein (“Crp”).
 11. The vaccine of claim 1 wherein the antigen is a polypeptide produced by a pathogen.
 12. The vaccine of claim 11 wherein the pathogen is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus group B, Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Staphylococcus spp., Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Erysipelothrix rhusiopathiae, Bacillus anthracis, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Clostridium tetani, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Corynebacterium diphtheriae, and Mycoplasma spp.
 13. The vaccine of claim 12 wherein the pathogen is Streptococcus pneumoniae.
 14. The vaccine of claim 1 wherein the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 15. The vaccine of claim 13 wherein the antigen is a pneumococcal surface protein A (“PspA”).
 16. The vaccine of claim 15 wherein the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 17. The vaccine of claim 16 wherein the first polynucleotide encodes a sequence as set forth in SEQ ID NO:1 and the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2.
 18. The vaccine of claim 17 wherein the strain of bacteria is a Salmonella enterica, which comprises (a) a mutation that renders the gene encoding cyclic AMP receptor protein inactive, (b) an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and (c) a plasmid, which comprises (i) the first polynucleotide, (ii) the second polynucleotide and (iii) a polynucleotide that encodes a functional Asd.
 19. The vaccine of claim 13 wherein the first polynucleotide and the second polynucleotide together comprise the sequence as set forth in SEQ ID NO:20.
 20. An immunogenic composition comprising a live attenuated strain of pathogenic gram negative bacteria, wherein (a) the strain of bacteria comprises a first polynucleotide that encodes an antigen, (b) the first polynucleotide is operably linked to a second polynucleotide that encodes a secretion peptide, (c) the antigen is from a source that is different than the live attenuated strain of bacteria, and (d) the immunogenic composition elicits a Th2-type immune response in a vertebrate.
 21. The immunogenic composition of claim 20 further comprising a balanced-lethal host-vector system.
 22. The immunogenic composition of claim 21 further comprising an environmental limitation viability system.
 23. The immunogenic composition of claim 21 wherein the live attenuated strain of bacteria comprises an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and a plasmid, wherein the plasmid comprises a polynucleotide that encodes a functional Asd and the first and second polynucleotides.
 24. The immunogenic composition of claim 20 wherein the strain of bacteria is a member of a group of bacteria selected from the group consisting of Enterobacteriaceae, Vibrionaceae, Francisellaceae, Legionallales, Pseudomonadacea and Pasteurellaceae.
 25. The immunogenic composition of claim 24 wherein the strain of bacteria is a member of the Enterobacteriaceae group.
 26. The immunogenic composition of claim 25 wherein the strain of bacteria is selected from the group consisting of Salmonella, Escherichia, Shigella and Yersinia.
 27. The immunogenic composition of claim 26 wherein the strain of bacteria is a Salmonella enterica.
 28. The immunogenic composition of claim 27 wherein the Salmonella enterica comprises an attenuating mutation in a gene encoding a protein selected from the group consisting of cyclic AMP receptor protein (“Crp”), adenylate cyclase (“Cya”), aspartate β-semialdehyde dehydrogenase (“Asd”) and DNA adenine methylase (“Dam”).
 29. The immunogenic composition of claim 28 wherein the protein is cyclic AMP receptor protein (“Crp”).
 30. The immunogenic composition of claim 20 wherein the antigen is a polypeptide produced by a pathogen.
 31. The immunogenic composition of claim 30 wherein the pathogen is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus group B, Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Staphylococcus spp., Erysipelothrix rhusiopathiae, Bacillus anthracis, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Clostridium tetani, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Corynebacterium diphtheriae, and Mycoplasma spp.
 32. The immunogenic composition of claim 31 wherein the pathogen is Streptococcus pneumoniae.
 33. The immunogenic composition of claim 20 wherein the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 34. The immunogenic composition of claim 32 wherein the antigen is a pneumococcal surface protein A (“PspA”).
 35. The immunogenic composition of claim 34 wherein the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 36. The immunogenic composition of claim 35 wherein the first polynucleotide encodes a sequence as set forth in SEQ ID NO:1 and the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2.
 37. The immunogenic composition of claim 36 wherein the strain of bacteria is a Salmonella enterica, which comprises (a) a mutation that renders the gene encoding cyclic AMP receptor protein inactive, (b) an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and (c) a plasmid, which comprises (i) the first polynucleotide, (ii) the second polynucleotide and (iii) a polynucleotide that encodes a functional Asd.
 38. The immunogenic composition of claim 31 wherein the first polynucleotide and the second polynucleotide together comprise the sequence as set forth in SEQ ID NO:20.
 39. A method of eliciting an immune response in a vertebrate, the method comprising administering a live attenuated strain of gram negative bacteria to said vertebrate, wherein (a) the strain of bacteria comprises a first polynucleotide that encodes an antigen, (b) the first polynucleotide is operably linked to a second polynucleotide that encodes a secretion peptide, (c) the antigen is from a source that is different than the live attenuated strain of bacteria, (d) the antigen is secreted from the strain of bacteria, and (e) the vertebrate produces IgG1 antibodies that specifically bind to the antigen.
 40. The method of claim 39 wherein the strain of bacteria comprises a balanced-lethal host-vector system.
 41. The method of claim 40 wherein the strain of bacteria comprises an environmental limitation viability system.
 42. The method of claim 40 wherein the live attenuated strain of bacteria comprises an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and a plasmid, wherein the plasmid comprises a polynucleotide that encodes a functional Asd and the first and second polynucleotides.
 43. The method of claim 39 wherein the strain of bacteria is a member of a group of bacteria selected from the group consisting of Enterobacteriaceae, Vibrionaceae, Francisellaceae, Legionallales, Pseudomonadacea and Pasteurellaceae.
 44. The method of claim 43 wherein the strain of bacteria is a member of the Enterobacteriaceae group.
 45. The method of claim 44 wherein the strain of bacteria is selected from the group consisting of Salmonella, Escherichia, Shigella and Yersinia.
 46. The method of claim 45 wherein the strain of bacteria is a Salmonella enterica.
 47. The method of claim 46 wherein the Salmonella enterica comprises an attenuating mutation in a gene encoding a protein selected from the group consisting of cyclic AMP receptor protein (“Crp”), adenylate cyclase (“Cya”), aspartate β-semialdehyde dehydrogenase (“Asd”) and DNA adenine methylase (“Dam”).
 48. The method of claim 47 wherein the protein is cyclic AMP receptor protein (“Crp”).
 49. The method of claim 39 wherein the antigen is a polypeptide produced by a pathogen.
 50. The method of claim 49 wherein the pathogen is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus group B, Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Staphylococcus spp., Erysipelothrix rhusiopathiae, Bacillus anthracis, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Clostridium tetani, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Corynebacterium diphtheriae, and Mycoplasma spp.
 51. The method of claim 50 wherein the pathogen is Streptococcus pneumoniae.
 52. The method of claim 39 wherein the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 53. The method of claim 51 wherein the antigen is a pneumococcal surface protein A (“PspA”).
 54. The method of claim 53 wherein the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 55. The method of claim 54 wherein the first polynucleotide encodes a sequence as set forth in SEQ ID NO:1 and the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2.
 56. The method of claim 55 wherein the strain of bacteria is a Salmonella enterica, which comprises (a) a mutation that renders the gene encoding cyclic AMP receptor protein inactive, (b) an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and (c) a plasmid, which comprises (i) the first polynucleotide, (ii) the second polynucleotide and (iii) a polynucleotide that encodes a functional Asd.
 57. The method of claim 51 wherein the first polynucleotide and the second polynucleotide together comprise the sequence as set forth in SEQ ID NO:20.
 58. The method of claim 56 wherein the vertebrate is a human and the strain of bacteria is administered orally.
 59. A live attenuated strain of pathogenic bacteria comprising a first polynucleotide that encodes an antigen, wherein (a) the first polynucleotide is operably linked to a second polynucleotide that encodes a secretion peptide, (b) the antigen is from a source that is different than the live attenuated strain of bacteria, (e) the antigen is secreted from the live attenuated strain of pathogenic bacteria, and (f) the pathogenic bacteria is an Enterobacteriaceae.
 60. The live attenuated strain of pathogenic bacteria of claim 59 further comprising a balanced-lethal host-vector system.
 61. The live attenuated strain of pathogenic bacteria of claim 60 further comprising an environmental limitation viability system.
 62. The live attenuated strain of pathogenic bacteria of claim 60 wherein the live attenuated strain of bacteria comprises an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and a plasmid, wherein the plasmid comprises a polynucleotide that encodes a functional Asd and the first and second polynucleotides.
 63. The live attenuated strain of pathogenic bacteria of claim 59 wherein the strain of bacteria is selected from the list consisting of Salmonella, Shigella, Escherichia and Yersinia.
 64. The live attenuated strain of pathogenic bacteria of claim 63 wherein the strain of bacteria is a Salmonella enterica.
 65. The live attenuated strain of pathogenic bacteria of claim 64 wherein the Salmonella enterica contains a mutation that renders the gene encoding cyclic AMP receptor protein inactive.
 66. The live attenuated strain of pathogenic bacteria of claim 59 wherein the antigen is a polypeptide produced by a pathogen.
 67. The live attenuated strain of pathogenic bacteria of claim 66 wherein the pathogen is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus group B, Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Staphylococcus spp., Erysipelothrix rhusiopathiae, Bacillus anthracis, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Clostridium tetani, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Corynebacterium diphtheriae, and Mycoplasma spp.
 68. The live attenuated strain of pathogenic bacteria of claim 67 wherein the pathogen is Streptococcus pneumoniae.
 69. The live attenuated strain of pathogenic bacteria of claim 59 wherein the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 70. The live attenuated strain of pathogenic bacteria of claim 68 wherein the antigen is a pneumococcal surface protein A (“PspA”).
 71. The live attenuated strain of pathogenic bacteria of claim 70 wherein the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 72. The live attenuated strain of pathogenic bacteria of claim 71 wherein the first polynucleotide encodes a sequence as set forth in SEQ ID NO:1 and the second polynucleotide encodes a sequence as set forth in SEQ ID NO:2.
 73. The live attenuated strain of pathogenic bacteria of claim 72 wherein the strain of bacteria is a Salmonella enterica, which comprises (a) a mutation that renders the gene encoding cyclic AMP receptor protein inactive, (b) an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and (c) a plasmid, which comprises (i) the first polynucleotide, (ii) the second polynucleotide and (iii) a polynucleotide that encodes a functional Asd.
 74. An immunogenic composition comprising a live attenuated strain of bacteria, wherein the strain of bacteria is an Enterobacteriaceae, which comprises a polynucleotide that encodes an antigen, wherein (i) the antigen is secreted from the cell, and (ii) the antigen is a polypeptide produced by a pathogen that is different than the live attenuated strain of bacteria.
 75. The immunogenic composition of claim 74 wherein the strain of bacteria is a Salmonella.
 76. The immunogenic composition of claim 75 wherein the pathogen is a Streptococcus bacteria.
 77. The immunogenic composition of claim 76 wherein the antigen is a pneumococcal surface protein A (“PspA”).
 78. The immunogenic composition of claim 77 wherein the strain of bacteria is Salmonella enterica.
 79. The immunogenic composition of claim 78 wherein the immunogenic composition elicits the production in a vertebrate of IgG1 antibodies that bind to the pneumococcal surface protein A.
 80. An immunogenic composition comprising a live attenuated strain of pathogenic gram negative bacteria, wherein (a) the strain of bacteria comprises (i) a first polynucleotide that encodes a first antigen, (ii) a second polynucleotide that encodes a second antigen and (iii) a third polynucleotide that encodes a secretion peptide, (b) the first antigen is from a serotype of a pathogen that is different from the live attenuated strain of pathogenic gram negative bacteria, (c) the second antigen is from a different serotype of the same pathogen from which the first antigen is derived and (d) the immunogenic composition elicits a Th2-type immune response in a vertebrate.
 81. The immunogenic composition of claim 80 further comprising a balanced-lethal host-vector system.
 82. The immunogenic composition of claim 81 further comprising an environmental limitation viability system.
 83. The immunogenic composition of claim 81 wherein the live attenuated strain of bacteria comprises an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and a plasmid, wherein the plasmid comprises a polynucleotide that encodes a functional Asd and the first, second and third polynucleotides.
 84. The immunogenic composition of claim 80 wherein the strain of bacteria is a member of a group of bacteria selected from the group consisting of Enterobacteriaceae, Vibrionaceae, Francisellaceae, Legionallales, Pseudomonadacea and Pasteurellaceae.
 85. The immunogenic composition of claim 84 wherein the strain of bacteria is a member of the Enterobacteriaceae group.
 86. The immunogenic composition of claim 85 wherein the strain of bacteria is selected from the group consisting of Salmonella, Escherichia, Shigella and Yersinia.
 87. The immunogenic composition of claim 86 wherein the strain of bacteria is a Salmonella enterica.
 88. The immunogenic composition of claim 87 wherein the Salmonella enterica comprises an attenuating mutation in a gene encoding a protein selected from the group consisting of cyclic AMP receptor protein (“Crp”), adenylate cyclase (“Cya”), aspartate β-semialdehyde dehydrogenase (“Asd”) and DNA adenine methylase (“Dam”).
 89. The immunogenic composition of claim 88 wherein the protein is cyclic AMP receptor protein (“Crp”).
 90. The immunogenic composition of claim 89 wherein the pathogen is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus group B, Streptococcus mutans, Streptococcus sobrinus, Streptococcus equi, Staphylococcus spp., Erysipelothrix rhusiopathiae, Bacillus anthracis, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Clostridium tetani, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Corynebacterium diphtheriae, and Mycoplasma spp.
 91. The immunogenic composition of claim 90 wherein the pathogen is Streptococcus pneumoniae.
 92. The immunogenic composition of claim 80 wherein the third polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 93. The immunogenic composition of claim 91 wherein the first antigen is a pneumococcal surface protein A (“PspA”).
 94. The immunogenic composition of claim 93 wherein the third polynucleotide encodes a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
 95. The immunogenic composition of claim 94 wherein the first polynucleotide encodes a sequence as set forth in SEQ ID NO:1, the second polynucleotide encodes a pneumococcal surface protein A from Streptococcus pneumoniae strain Rx1, and the third polynucleotide encodes a sequence as set forth in SEQ ID NO:2.
 96. The immunogenic composition of claim 95 wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide together comprise the sequence as set forth in SEQ ID NO:20.
 97. The immunogenic composition of claim 96 wherein the strain of bacteria is a Salmonella enterica, which further comprises (a) a mutation that renders the gene encoding cyclic AMP receptor protein inactive, (b) an inactivating mutation in the chromosomal gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and (c) a plasmid, which comprises (i) the first polynucleotide, (ii) the second polynucleotides, (iii) the third polynucleotides and (iv) a polynucleotide that encodes a functional Asd. 